Compartmentalisation of second messenger signalling pathways

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ScienceDirect Compartmentalisation of second messenger signalling pathways Kristie McCormick and George S Baillie The ability of a cell to transform an extracellular stimulus into a downstream event that directs specific physiological outcomes, requires the orchestrated, spatial and temporal response of many signalling proteins. The notion of compartmentalised signalling pathways was popularised in the 1980s by Brunton and colleagues, with their discovery that spatially segregated cAMP directs a variety of signalling responses in cardiomyocytes. It is now understood that compartmentalisation is a common mechanism used by all cells to ensure the interaction of signalling ‘second messenger’ molecules with localised ‘pools’ of appropriate effector proteins. In this way, the cell can elicit differential cellular responses by using a single, freely diffusible, molecular species. Recently, the compartmentalisation schemes employed by signalling systems involving cyclic nucleotides, calcium and nitric oxide have been elucidated and as a result, the varied range of functional consequences underpinned by such strategies can be better appreciated. Addresses Institute of Cardiovascular and Medical Sciences, CMVLS, Wolfson-Link Building, University of Glasgow, Glasgow G12 8QQ, UK Corresponding author: Baillie, George S ([email protected])

Current Opinion in Genetics & Development 2014, 27:20–25 This review comes from a themed issue on Developmental mechanisms, patterning and evolution Edited by Lee A Niswander and Lori Sussel

0959-437X/$ – see front matter, # 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.gde.2014.02.001

Introduction In order to translate the events occurring at the cell surface into an intracellular response, the cell enables specific molecules, so-called ‘second messengers’, as chemical relays to engage the signalling machinery within the cytoplasm. In short, cellular stimulation via cell surface receptors induces (often at the plasma membrane) transient production of signalling molecules that ‘find’ and bind to an appropriate effector protein. This action serves to initiate a signal cascade, which both amplifies the signal and directs it towards the alteration of a specific cellular process in response to the initial environmental threat or chemical cue on the outside of the cell. Despite the underlying mechanism being similar for each second messenger, the cellular responses are highly diverse, ranging from gene activation to cell division [1,2]. Current Opinion in Genetics & Development 2014, 27:20–25

A multiplicity of function also exists within each second messenger system, as a single chemical species may have multiple downstream effectors and regulate numerous physiological processes. Essentially, the cell achieves this feat by the spatio-temporal regulation, or compartmentalisation, of its second messengers to allow discrete pathways of localised signalling activity to occur. This review will discuss compartmentalisation with respect to four well-studied second messenger systems; cAMP, cGMP, calcium and nitric oxide (NO), concentrating on the way in which each system evokes its effects in a spatially restricted manner. The functional implications from recent groundbreaking studies will also be highlighted and discussed.

Compartmentalised cAMP signalling 30 ,50 -Cyclic adenosine monophosphate (cAMP) was the first second messenger to be discovered [3] and many of the concepts that we take for granted surrounding signal generation via effectors [4] and compartmentalisation [5] stem from seminal work on this molecule. Today, we understand that, when activated, a variety of different cell surface receptors coupled to Gas, educe equivalent increases in cAMP production that result in diverse cellular responses. This can only happen if the signalling ‘machinery’ for each process is spatially linked to the ‘pool’ of cAMP produced by adenylate cyclase in response to distinct receptors [6]. Such compartmentalisation allows anchored cAMP effectors such as Protein Kinase A (PKA) [7] and the exchange protein activated by cAMP (EPAC) [8] to sample cAMP transients and produce downstream signals when their activation threshold (in terms of cAMP concentration) is surpassed (Figure 1). The duration and strength of signals produced by cAMP effectors is heavily influenced by action of the only superfamily of enzymes that has evolved to degrade cAMP, the cAMP-phosphodiesterases (PDEs) [9]. The pivotal role of PDEs in shaping the spatial and temporal dimensions of cAMP ‘pulses’ produced in response to receptor activation was realised following the invention of a number of cAMP reporters that allowed real-time visualization of cAMP dynamics within cellular micro-domains [10]. These probes revealed spatially restricted cAMP gradients that were tailored by discretely anchored PDE populations to allow activation of small subsets of cAMP effectors. In effect, the PDEs acted as local cyclic nucleotide ‘sinks’ allowing ‘hotspots’ of cAMP to accumulate in areas of the cell that were devoid of PDEs (Figure 1). This action underpinned receptor specificity of action, as co-treatment with a non-specific PDE inhibitor and receptor agonist, allowed uniform, global increases in cAMP and concomitant activation of all cAMP effectors [11]. www.sciencedirect.com

Compartmentalised signalling McCormick and Baillie 21

Figure 1

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Current Opinion in Genetics & Development

Compartmentalisation of cAMP signalling. (A) The G-protein subunit Gas is released following receptor activation by the appropriate ligand. (B) The subunit binds adenylate cyclase (AC) to trigger production of cAMP. (C) Phosphodiesterases (PDEs) shape the cAMP gradient ensuring the specificity of signals emanating from defined receptors. (D) cAMP effectors, such as PKA and EPAC, that are in close proximity to a PDE ‘pool’ cannot be activated unless the associated PDE is ‘swamped’ by the cAMP produced in response to activation of the receptor. (E) The threshold of activation for cAMP effectors not in the vicinity of PDEs is more easily reached. These enzymes drive downstream signalling events that orchestrate physiological changes in response to the extracellular stimulus detected by cell-surface receptors.

Although the fundamental roles that cAMP-PDEs play in tailoring compartmentalised cAMP signals have been supported by studies using pharmacological inhibition of PDEs [12], siRNA silencing [13], transfection of dominant negative PDE isoforms [14], knockout mice [15], mathematical modelling [16] and peptide interference [17], other concepts such as local changes in viscosity and structural impediments to free cAMP diffusion have been offered as explanations for selective activation of cAMP effectors following receptor activation [18].

stimulation [19]. The stimuli for activation, however, are distinct, and include peptide hormones and nitric oxide. Peptide hormones typically activate membrane-bound GC (pGC), whereas nitric oxide activates the soluble form (sGC). Downstream effectors of this pathway include cyclic nucleotide gated cation channels and cGMP dependent protein kinase (PKG) (Figure 2). Hydrolysis of cGMP is catalysed by cGMP-PDEs. As with the cAMP system, compartmentalisation of enzymes that produce, are activated by and degrade cGMP is key to the signalling fidelity of this system [20].

Compartmentalised cGMP signalling Cyclic GMP is a similar second messenger to cAMP, in that both are cyclic nucleotides with comparable mechanisms of signalling. In an analogous manner to cAMP, cGMP is synthesised from GTP by the enzyme guanylate cyclase (GC) in response to G-protein coupled receptor www.sciencedirect.com

The action of cGMP-PDEs is pivotal for the regulation of spatial and temporal cGMP dynamics, with PDE5 and PDE2 having major roles. The PDE family member with the greatest influence appears to be determined by the cGMP source being considered. PDE5 and PDE2 limit Current Opinion in Genetics & Development 2014, 27:20–25

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Figure 2

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Downstream events: e.g. regulation of cardiac contraction Current Opinion in Genetics & Development

Nitric oxide generation and signalling. Nitric oxide (NO) produced by nitric oxide synthase (NOS) drives downstream signalling by (A) promoting the modification of other signalling proteins or (B) via the activation of guanylate cyclase in the cytoplasm (sGC) or at the membrane (pGC). (C) sGC can result in events such as the spatially restricted activation of ERK MAP kinase cascade following the S-nitrosylation (SNO) of membrane-bound Ras [32]. pGC results in the production of cGMP, which activates (D) PKG and (E) cyclic nucleotide gated ion channels in order to trigger a variety of physiological alterations, including changes in cardiac contraction [24].

sGC and pGC-derived cGMP signals, respectively [21]. Functionally, this has had implications in understanding the regulation of cardiac contractility, as localised PDE5 activity is required to underpin specific sGC action (via PKG) in promoting b-adrenergic (b-AR) stimulation [22]. Indeed, pharmacological inhibition of this PDE5 pool promoted global cGMP increases that actually resulted in a retardation of b-AR signalling. In contrast, cGMP increases triggered by natriuretic peptide do not modulate b-AR responsiveness, once again suggesting that these different cGMP pools are relevant only in their defined location [23]. This also holds true for cGMP gradients, which are shaped by the action of PDE2. It appears that PDE2 modulates cardiac function through its localisation to lipid rafts in the plasma membrane. Such localisation, along with adenylate cyclase, b-AR, and nitric oxide synthase (NOS) is thought to underpin its coupling to b3-AR-dependent NOS/cGMP activation of PDE and subsequent cAMP hydrolysis [24]. Another level of compartmentalised cGMP regulation depends on the PKG mediated phosphorylation of Current Opinion in Genetics & Development 2014, 27:20–25

PDE and guanylate cyclase. A recent study showed that PKG activation limits the accumulation and intracellular diffusion of NO-stimulated, sGC-derived cGMP through the phosphorylation of PDE5 (an action which increases PDE5 hydrolytic activity) [25]. This mechanism is not surprising, considering the parallels with a similar ‘feedback’ loop, which involves PDE3 and PDE4 activation following phosphorylation by PKA. However, at the sarcolemmal membrane, this action can be countered by a PKG-dependent increase in ANP (natriuretic peptide receptor)-stimulated, pGC-derived, cGMP production, where PKG phosphorylates pGC directly, to trigger its activation. So in effect, PKG regulates both membrane and cytosolic cGMP dynamics but in opposite directions.

Compartmentalised NO signalling As NO is a gas, it cannot be considered as a typical second messenger, however it is an important signalling molecule involved in the regulation of a myriad of cellular processes ranging from vasodilation to respiration [26,27]. Produced by the action of NOS, NO is a lipophillic and short-lived www.sciencedirect.com

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messenger, which mediates its responses through one of two mechanisms; cGMP production via sGC activation or chemical modification of proteins via reactive nitrogen species (e.g. S-nitrosylation) (Figure 2). Compartmentalisation of this highly diffusible molecule is conferred by the subcellular localisation of NO sources. In terms of S-nitrosylation, it has been noted that membrane localisation of NOS is one of the ways in which this signalling system is compartmentalised. eNOS, for example, traffics between the plasma membrane and Golgi apparatus in order to ensure proximity to appropriate target proteins [28]. Another level of regulation is conferred by the fact that Golgi and plasma membranelocated eNOS enzymes have differing thresholds of activation as well as NO production rates (less eNOS is released from Golgi), thus, the locality of the enzyme is doubly important in governing spatial and temporal response to NO [29,30]. Indeed, a recent study concluded that the greatest influence in the determination of functional outcomes for differently located NOS isoforms was the amount of NO produced [31]. The implications of NO compartmentalisation on cellular functioning are only now becoming realised. Indeed, it has been found recently that localised NO production ensures Ras activation is limited to the plasma membrane [32] (Figure 2). The study in question reported that bradykinin treatment in endothelial cells, selectively stimulates H-Ras at the plasma membrane via S-nitrosylation, an action which is independent of Src kinase action or EGFR activation. As only the membrane bound pool of Ras was activated, the authors suggested that the compartmentalisation of the subsequent downstream signal was underpinned by the restricted production of NO in localised areas. These findings are in agreement with other descriptions of NO sources, which have been restricted to defined organelles e.g. Golgi, nucleus [33]. Specifically, it is thought that intracellular S-nitrosylation of substrates is limited to the primary site of eNOS localisation, and this allows fine control of spatial physiological changes, for example initiation of intracellular transport processes.

development to muscle contraction [37,38]. Regulation of calcium compartmentalisation differs from that seen with cyclic nucleotides, as there is no enzymatic degradation of the molecule, so spatial/temporal control of Ca2+ concentration depends on only two parameters, site of release/ uptake and diffusion rate. Indeed, calcium can induce its own release from intracellular stores (Ca2+ induced Ca2+ release), an ability that can rapidly amplify cellular responses to small amounts of this second messenger. It can also be rapidly pumped back into calcium stores or sequestered by Ca2+ binding proteins [39]. As the diffusion rate of calcium is relatively slow (one tenth of cAMP), Ca2+ ATPase pumps/exchangers, which act to move large amounts of Ca2+ to and from intracellular locations, play the most important role in Ca2+ compartmentalisation. Their importance to life is substantiated by the fact that there are nine different types of Ca2+ ATPase pumps/ exchangers belonging to three multi-gene families and they exhibit differences in tissue/cell distribution and modes of regulation [40]. In the resting cell, concentrations of Ca2+ within the cytoplasm are kept low (<100 nM) because high levels of free calcium are known to be toxic. During activation of Ca2+ regulated processes, levels can rapidly increase. As cells must ensure that such a massive Ca2+ influx does not result in global saturation and loss of selective target stimulation leading to a reduction in response specificity, large changes in Ca2+ concentration are represented by transient ‘spikes’, which can be further organized into regenerative waves [41]. The diffusion of these fleeting ‘spikes’ can be hindered by organelles (e.g. mitochondria or reticular ER), buffers or calcium binding proteins. Local Ca2+ concentration is ‘sampled’ by a variety of localised calcium effector proteins or lipids, which have varying affinities and activation thresholds (reviewed in [42]). It is these effectors, which ‘decode’ the information (amplitude, duration and frequency) within the calcium transient to ensure signalling fidelity.

Compartmentalised Ca2+ signalling

Recently, a novel cellular strategy has been discovered that augments compartmentalisation of Ca2+ signals. It has been shown that the cell can co-localise a variety of calcium effectors by sequestering them into caveolae micro-domains [43]. In addition, a recent study analysing intra-caveolar Ca2+ dynamics has suggested that caveolae can function as mini Ca2+ stores [44], which release calcium in an ATP-dependent manner. Practically, this has been suggested to aid in Ca2+ wave initiation at caveolin-rich edges via sensitisation of ER-localised IP3 receptors. Such a strategy constitutes a novel way to ‘fine-tune’ the temporal organization of Ca2+ signals in a small, well defined area of the cell.

In comparison to its fellow intracellular messengers, calcium is arguably the most versatile and widely utilised. Functionally, calcium orchestrates a plethora of physiological and developmental processes, ranging from sperm

Other novel functional correlates of compartmentalised calcium signalling include the discovery that localised Ca2+ transients in dendritic branches act as spatiotemporal

The role of NO signalling in development is only beginning to be understood, however it is clear that the influence of NO in this regard is widespread, having roles in morphogenesis in plants [34], brain development [35] and the development of sexual organs [36]. The importance of compartmentalisation to these processes should be an avenue of future research.

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signals to initiate sensory neuron pruning in Drosophila [45]. Specifically, the Ca2+-activated protease calpain, was observed to function downstream of these transients, in order to facilitate dendrite pruning that is required to shape neural circuits. Calcium compartmentalisation has also been key to a unique form of head movement in C. elegans, where the muscarinic acetylcholine receptor GAR3 acts to mobilise calcium in a set of interneurons [46]. Local calcium release in this manner has been shown to corral axonal activity to encode head movement, an action that is distinct from normal synchronised locomotory behaviour.

Conclusions In summation, we have discussed the attributes of four different but ubiquitous ‘second messenger’-signalling systems, which underpin specific cellular responses to extra-cellular events. Transient, diffusion of chemical messenger waves can only lead to meaningful physiological responses following the orchestrated generation and degradation of these signals in three dimensions in combination with the appropriate activation of effector proteins. Each system has evolved its own molecular mechanisms and feedback systems to provide such a level of control that extends to discrete areas of a cell. Intriguingly, each of the four systems we have discussed can impinge on each other via various points of ‘cross-talk’ between the pathways. Unfortunately, a discussion of this cross-talk is outside the scope of the current review.

Acknowledgements Work from the Baillie Lab mentioned in this review was funded by the Medical Research Council UK (MR/J0074112/1) and Heart Research UK (RG2610/12/14).

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1.

Abel S et al.: Bi-modal distribution of the second messenger cdi-GMP controls cell fate and asymmetry during the caulobacter cell cycle. PLoS Genet 2013, 9(9):e1003744.

2.

Lalli E, Sassone-Corsi P: Signal transduction and gene regulation: the nuclear response to cAMP. J Biol Chem 1994, 269(26):17359-17362.

3.

Rall TW, Sutherland EW: Formation of a cyclic adenine ribonucleotide by tissue particles. J Biol Chem 1958, 232(2):1065-1076.

4.

Walsh DA, Perkins JP, Krebs EG: An adenosine 3’,5’monophosphate-dependant protein kinase from rabbit skeletal muscle. J Biol Chem 1968, 243(13):3763-3765.

5.

Buxton IL, Brunton LL: Compartments of cyclic AMP and protein kinase in mammalian cardiomyocytes. J Biol Chem 1983, 258(17):10233-10239.

6.

Baillie GS: Compartmentalized signalling: spatial regulation of cAMP by the action of compartmentalized phosphodiesterases. FEBS J 2009, 276(7):1790-1799.

7.

Wong W, Scott JD: AKAP signalling complexes: focal points in space and time. Nat Rev. Mol Cell Biol 2004, 5(12):959-970.

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8.

Gloerich M, Bos JL: Epac: defining a new mechanism for cAMP action. Annu Rev Pharmacol Toxicol 2010, 50:355-375.

9.

Conti M, Beavo J: Biochemistry and physiology of cyclic nucleotide phosphodiesterases: essential components in cyclic nucleotide signaling. Annu Rev Biochem 2007, 76:481-511.

10. Zaccolo M: Use of chimeric fluorescent proteins and fluorescence resonance energy transfer to monitor cellular responses. Circulat Res 2004, 94(7):866-873. 11. Zaccolo M, Pozzan T: Discrete microdomains with high concentration of cAMP in stimulated rat neonatal cardiac myocytes. Science 2002, 295(5560):1711-1715. 12. Vecsey CG et al.: Sleep deprivation impairs cAMP signalling in the hippocampus. Nature 2009, 461(7267):1122-1125. 13. Lynch MJ et al.: RNA silencing identifies PDE4D5 as the functionally relevant cAMP phosphodiesterase interacting with beta arrestin to control the protein kinase A/AKAP79mediated switching of the beta2-adrenergic receptor to activation of ERK in HEK293B2 cells. J Biol Chem 2005, 280(39):33178-33189. 14. Brown KM et al.: Phosphodiesterase-8A binds to and regulates Raf-1 kinase. Proc Natl Acad Sci U S A 2013, 110(16):E1533E1542. 15. Lehnart SE et al.: Phosphodiesterase 4D deficiency in the ryanodine-receptor complex promotes heart failure and arrhythmias. Cell 2005, 123(1):25-35. 16. Chen W, Levine H, Rappel WJ: A mathematical analysis of second messenger compartmentalization. Phys Biol 2008, 5(4):046006. 17. Lee LC, Maurice DH, Baillie GS: Targeting protein–protein  interactions within the cyclic AMP signaling system as a therapeutic strategy for cardiovascular disease. Future Med Chem 2013, 5(4):451-464. This paper proposes the concept that displacement of cAMP signalling proteins from their intra-cellular site of action may be a novel therapeutic route to prevent the ‘off-target’ effects following active site directed inhibition of cAMP effectors and/or phosphodiesterases. 18. Feinstein WP, Zhu B, Leavesley SJ, Sayner SL, Rich TC: Assessment of cellular mechanisms contributing to cAMP compartmentalization in pulmonary microvascular endothelial cells. Am J Physiol Cell Physiol 2012, 302(6):C839C852. 19. Kots AY, Martin E, Sharina IG, Murad F: A short history of cGMP, guanylyl cyclases, and cGMP-dependent protein kinases. Handbook Exp Pharmacol 2009, 191:1-14. 20. Nikolaev VO, Lohse MJ: Novel techniques for real-time monitoring of cGMP in living cells. Handbook Exp Pharmacol 2009, 191:229-243. 21. Castro LR, Verde I, Cooper DM, Fischmeister R: Cyclic guanosine monophosphate compartmentation in rat cardiac myocytes. Circulation 2006, 113(18):2221-2228. 22. Takimoto E et al.: Compartmentalization of cardiac betaadrenergic inotropy modulation by phosphodiesterase type 5. Circulation 2007, 115(16):2159-2167. 23. Takimoto E et al.: cGMP catabolism by phosphodiesterase 5A regulates cardiac adrenergic stimulation by NOS3-dependent mechanism. Circulat Res 2005, 96(1):100-109. 24. Mongillo M et al.: Compartmentalized phosphodiesterase-2 activity blunts beta-adrenergic cardiac inotropy via an NO/cGMP-dependent pathway. Circulat Res 2006, 98(2):226-234. 25. Castro LR, Schittl J, Fischmeister R: Feedback control through cGMP-dependent protein kinase contributes to differential regulation and compartmentation of cGMP in rat cardiac myocytes. Circulat Res 2010, 107(10):1232-1240. 26. Palmer RM, Ferrige AG, Moncada S: Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987, 327(6122):524-526. www.sciencedirect.com

Compartmentalised signalling McCormick and Baillie 25

27. Beltran B, Orsi A, Clementi E, Moncada S: Oxidative stress and S-nitrosylation of proteins in cells. Br J Pharmacol 2000, 129(5):953-960. 28. Martinez-Ruiz A, Lamas S: Signalling by NO-induced protein Snitrosylation and S-glutathionylation: convergences and divergences. Cardiovasc Res 2007, 75(2):220-228. 29. Zhang Q et al.: Functional relevance of Golgi- and plasma membrane-localized endothelial NO synthase in reconstituted endothelial cells. Arteriosclerosis Thrombosis Vascular Biol 2006, 26(5):1015-1021. 30. Fulton D et al.: Targeting of endothelial nitric-oxide synthase to the cytoplasmic face of the Golgi complex or plasma membrane regulates Akt- versus calcium-dependent mechanisms for nitric oxide release. J Biol Chem 2004, 279(29):30349-30357. 31. Qian J et al.: Role of local production of endothelium-derived nitric oxide on cGMP signaling and S-nitrosylation. Am J Physiol Heart Circulatory Physiol 2010, 298(1):H112-H118. 32. Batista WL et al.: S-nitrosoglutathione and endothelial nitric  oxide synthase-derived nitric oxide regulate compartmentalized ras S-nitrosylation and stimulate cell proliferation. Antioxidants Redox Signal 2013, 18(3):221-238. Intruiging report that NO-mediated activation of RAS in different cellular locations acts to differentially activate downstream signalling pathways. This novel finding underpins the notion that compartmentalisation of NO is essential to maintain signalling specificity 33. Iwakiri Y et al.: Nitric oxide synthase generates nitric oxide locally to regulate compartmentalized protein S-nitrosylation and protein trafficking. Proc Natl Acad Sci U S A 2006, 103(52):19777-19782. 34. Hebelstrup KH, Shah JK, Igamberdiev AU: The role of nitric oxide and hemoglobin in plant development and morphogenesis. Physiol Plant 2013, 148(4):457-469. 35. Contestabile A: Role of nitric oxide in cerebellar development and function: focus on granule neurons. Cerebellum 2012, 11(1):50-61. 36. Auharek SA, Lara NL, Avelar GF, Sharpe RM, Franca LR: Effects of inducible nitric oxide synthase (iNOS) deficiency in mice on

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Sertoli cell proliferation and perinatal testis development. Int J Androl 2012, 35(5):741-751. 37. Ebashi S, Endo M: Calcium ion and muscle contraction. Progr Biophys Mol Biol 1968, 18:123-183. 38. Darszon A, Nishigaki T, Beltran C, Trevino CL: Calcium channels in the development, maturation, and function of spermatozoa. Physiol Rev 2011, 91(4):1305-1355. 39. Carafoli E, Santella L, Branca D, Brini M: Generation, control, and processing of cellular calcium signals. Crit Rev Biochem Mol Biol 2001, 36(2):107-260. 40. Brini M, Cali T, Ottolini D, Carafoli E: Calcium pumps: why so many? Comprehensive Physiol 2012, 2(2):1045-1060. 41. Rey O, Young SH, Jacamo R, Moyer MP, Rozengurt E: Extracellular calcium sensing receptor stimulation in human colonic epithelial cells induces intracellular calcium oscillations and proliferation inhibition. J Cell Physiol 2010, 225(1):73-83. 42. Clapham DE: Calcium signaling. Cell 2007, 131(6):1047-1058. 43. Pani B, Singh BB: Lipid rafts/caveolae as microdomains of calcium signaling. Cell Calcium 2009, 45(6):625-633. 44. Isshiki M, Nishimoto M, Mizuno R, Fujita T: FRET-based sensor  analysis reveals caveolae are spatially distinct Ca stores in endothelial cells. Cell Calcium 2013. First indication that caveolae can act as mini calcium stores. This discovery uncovers a new ‘level’ at which compartmentalisation of calcium can regulate cell signalling events. 45. Kanamori T et al.: Compartmentalized calcium transients  trigger dendrite pruning in Drosophila sensory neurons. Science 2013, 340(6139):1475-1478. This report proposes that inappropriate dendrite branches are ‘tagged’ for destruction by highly compartmentalised calcium ‘spikes’. As dendrtite pruning is essential to sculpt neural circuits, this represents a new function for spatially restricted calcium flux. 46. Hendricks M, Ha H, Maffey N, Zhang Y: Compartmentalized calcium dynamics in a C. elegans interneuron encode head movement. Nature 2012, 487(7405):99-103.

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