Intercellular Ca2 Research Articles

Abstract 5248: Tunneling nanotubes connect melanoma cells and mediate transfer of mitochondria and GRP78.

Abstract Background: Recently, exciting reports have outlined the existence and function of very thin, membranous conduits between cells that span significant distances, termed tunneling nanotubes (TnTs). TnTs have been observed in neoplastic and normal cells, and may transport soluble proteins, such as P-glycoprotein (MDR1), lipids, and whole organelles, including mitochondria and Golgi vesicles, between cells. Furthermore, TnTs mediate intercellular Ca2+ signals and transmit electrical potentials. However, their functional characterization remains incomplete. Objectives: Our objective is to describe TnT formation and function in melanoma cell lines and primary tumor samples. Methods: We describe the presence and composition of TnTs between cells in the melanoma cell lines SK-Mel-31, DM440, DM443, DM738, and DM598 in vitro with a combination of brightfield and fluorescent microscopy techniques. TnT formation is assessed in response to hypoxia, glucose stress, and low pH. Bulk intercellular transport of mitochondria is quantified via flow cytometry using MitotrackerTM dyes, and GFP-GRP78 fusion proteins are used for studies of protein transport. Additional flow cytometric studies include JC-1 staining to measure mitochondrial membrane potential, annexin V binding to measure apoptosis, and 7-AAD exclusion for live/dead staining. Fluorescent ATP assays are used to measure general metabolic capacity, and confocal microscopy of primary melanoma specimens is performed using melanosome-specific (P100) and dsDNA (H33342) stains. Results All tested cell lines formed TnT-like structures with variable F-actin and tubulin composition. Mitochondria and Golgi vesicles were visible within live and fixed TnTs, which were formed constitutively and were further induced by hypoxia, glucose stress, and low pH. In an actin-dependent process, cells rapidly transferred labeled mitochondria, achieving homogeneity within 24 h as measured by flow cytometry. Transfer of healthy mitochondria from unstressed cells significantly mitigated the acute toxicity of chemotherapeutically damaged cells. GRP78 was also rapidly transferred between cells, and the transfer of GRP78 from healthy donor cells rescued cells damaged by chemotherapeutics as measured by a decreased apoptotic rate. Conclusions: Here, we report for the first time, the characterization of TnTs between melanoma cells. Moreover, melanoma cells acquired primary and secondary chemotherapeutic resistance to alkylating agents melphalan and temozolomide and the BRAFV600E inhibitor, vemurafenib, via bulk intercellular transfer of functional mitochondria and the ER stress protein, GRP78. TnT inhibitors would represent a novel class of chemotherapeutics with implications for invasion, metastasis, and chemotherapeutic resistance. Citation Format: Gustaaf G. de Ridder, Rupa Ray, Salvatore V. Pizzo. Tunneling nanotubes connect melanoma cells and mediate transfer of mitochondria and GRP78. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5248. doi:10.1158/1538-7445.AM2013-5248 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Resveratrol Inhibits β-Amyloid-Induced Neuronal Apoptosis through Regulation of SIRT1-ROCK1 Signaling Pathway

Alzheimer’s disease (AD) is characterized by the accumulation of β-amyloid peptide (Aβ) and loss of neurons. Recently, a growing body of evidences have indicated that as a herbal compound naturally derived from grapes, resveratrol modulates the pathophysiology of AD, however, with a largely unclear mechanism. Therefore, we aimed to investigate the protection of resveratrol against the neurotoxicity of β-amyloid peptide 25–35 (Aβ25–35) and further explore its underlying mechanism in the present study. PC12 cells were injuried by Aβ25–35, and resveratrol at different concentrations was added into the culture medium. We observed that resveratrol increased cell viability through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) colorimetric assays. Flow cytometry indicated the reduction of cell apoptosis by resveratrol. Moreover, resveratrol also stabilized the intercellular Ca2+ homeostasis and attenuated Aβ25–35 neurotoxicity. Additionally, Aβ25–35-suppressed silent information regulator 1 (SIRT1) activity was significantly reversed by resveratrol, resulting in the downregulation of Rho-associated kinase 1 (ROCK1). Our results clearly revealed that resveratrol significantly protected PC12 cells and inhibited the β-amyloid-induced cell apoptosis through the upregulation of SIRT1. Moreover, as a downstream signal molecule, ROCK1 was negatively regulated by SIRT1. Taken together, our study demonstrated that SIRT1-ROCK1 pathway played a critical role in the pathomechanism of AD.

Human Articular Chondrocytes Express Multiple Gap Junction Proteins: Differential Expression of Connexins in Normal and Osteoarthritic Cartilage

Human Articular Chondrocytes Express Multiple Gap Junction Proteins: Differential Expression of Connexins in Normal and Osteoarthritic Cartilage

Intercellular Ca2+ wave propagation in human retinal pigment epithelium cells induced by mechanical stimulation

Ca2+ signaling is vitally important in cellular physiological processes and various drugs also affect Ca2+ signaling. Thus, knowledge of Ca2+ dynamics is important toward understanding cell biology, as well as the development of drug-testing assays. ARPE-19 cells are widely used for modeling human retinal pigment epithelium functions and drug-testing, but intercellular communication has not been assessed in these cells. In this study, we investigated intercellular Ca2+ communication induced by mechanical stimulation in ARPE-19 cells. An intercellular Ca2+ wave was induced in ARPE-19 monolayer by point mechanical stimulation of a single cell. Dynamic changes of intracellular Ca2+ concentration ([Ca2+]i) in the monolayer were tracked with fluorescence microscopy imaging using Ca2+-sensitive fluorescent dye fura-2 in presence and absence of extracellular Ca2+, after depletion of intracellular Ca2+ stores with thapsigargin, and after application of gap junction blocker α-glycyrrhetinic acid and P2-receptor blocker suramin. Normalized fluorescence values, reflecting amplitude of [Ca2+]i increase, and percentage of responsive cells were calculated to quantitatively characterize Ca2+ wave propagation. Mechanical stimulation of a single cell within a confluent monolayer of ARPE-19 cells initiated an increase in [Ca2+]i, which propagated to neighboring cells in a wave-like manner. Ca2+ wave propagated to up to 14 cell tiers in control conditions. The absence of extracellular Ca2+ reduced [Ca2+]i increase in the cells close to the site of mechanical stimulation, whereas the depletion of intracellular Ca2+ stores with thapsigargin blocked the wave spreading to distant cells. The gap junction blocker α-glycyrrhetinic acid reduced [Ca2+]i increase in the cell tiers close to the site of mechanical stimulation, indicating involvement of gap junctions in Ca2+ wave propagation. The P2-receptor blocker suramin reduced the percentage of responsive cells participating in Ca2+ wave spreading beyond the fourth cell tier, showing the necessity of P2-receptors for Ca2+ wave propagation. In disconnected, i.e., subconfluent, ARPE-19 cell clusters Ca2+ wave spreading was considerably less efficient compared to that in confluent ARPE-19 monolayer at the same distances. ARPE-19 cells showed repeatable and robust Ca2+ dynamics after mechanical stimulus. The ARPE-19 cells exhibited two different mechanisms of Ca2+ wave propagation dependent on the cell location: in the cells close to the site of mechanical stimulation the Ca2+ wave propagated mainly through gap junctions and required Ca2+ from both intracellular Ca2+ stores and extracellular media, while farther away the propagation was more dependent on the purinergic receptors and did not require extracellular Ca2+. The proposed method could provide a tool to assess the drug-induced changes in intercellular communication in in vitro assays in human retinal pigment epithelial cells.

Can triggered arrhythmias arise from propagation of calcium waves between cardiac myocytes

Intracellular Ca2+ overload can induce regenerative Ca2+ waves that activate inward current in cardiac myocytes, allowing the cell membrane to achieve threshold. The result is a triggered extrasystole that can, under the right conditions, lead to sustained triggered arrhythmias. In this review, we consider the issue of whether or not Ca2+ waves can travel between neighboring myocytes and if this intercellular Ca2+ diffusion can involve enough cells over a short enough period of time to actually induce triggered activity in the heart. This review is not intended to serve as an exhaustive review of the literature summarizing Ca2+ flux through cardiac gap junctions or of how Ca2+ waves move from cell to cell. Rather, it summarizes many of the pertinent experimental studies and considers their results in the theoretical context of whether or not the intercellular propagation of Ca2+ overload can contribute to triggered beats and arrhythmias in the intact heart.

Erratum

MicrocirculationVolume 20, Issue 1 p. 115-115 ErratumFree Access Erratum This article corrects the following: Coordination of Intercellular Ca2+ Signaling in Endothelial Cell Tubes of Mouse Resistance Arteries Matthew J. Socha, Timothy L. Domeier, Erik J. Behringer, Steven S. Segal, Volume 19Issue 8Microcirculation pages: 757-770 First Published online: November 20, 2012 First published: 09 January 2013 https://doi.org/10.1111/micc.12029AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat In Socha et al. (2012), the print issue published the citation reference incorrectly as “Microcirculation: 19: 749–756, 2012.” It should be “Microcirculation: 19: 757–770, 2012.” The online version has now been corrected. Reference Socha MJ, Domeier TL, Behringer EJ, Segal SS. Coordination of intercellular Ca2+ signaling in endothelial cell tubes of mouse resistance arteries. Microcirculation 19: 757– 770, 2012. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Volume20, Issue1January 2013Pages 115-115 ReferencesRelatedInformation

Ca Release Unit Heterogeneity and Entrainment of Ca Waves in Cardiac Myocytes

Calcium (Ca) is a ubiquitous second messenger regulating many biological functions. The elementary events of local Ca signaling are Ca sparks, which occur randomly in time and space, and integrate to produce global signaling events including intracellular and intercellular Ca waves and whole-cell Ca oscillations. In a recent study using a computational model of Ca signaling in a cardiac myocyte and then experimentally in mouse ventricular myocytes, we demonstrated that criticality is the underlying theoretical mechanism that governs the transition from local Ca sparks to global Ca waves, analogous to a second-order phase transition in thermodynamics. Theoretically, criticality predicts that wave initiation sites should occur randomly and uniformly in space. While this has been demonstrated experimentally at relatively low Ca loads, recent experiments suggest that at higher Ca loads certain regions of the cell dominate the wave initiation process, acting as pacemakers to entrain whole cell oscillations. Here we show that the formation of these pacemaking sites is still governed by the theory of criticality, however, heterogeneities of the ryanodine receptor clusters result in different firing frequencies, with the fastest regions tending to entrain the whole cell. We demonstrate that there is a critical size and relative degree of heterogeneity that must be reached for entrainment to occur, with the novel finding that the degree of entrainment depends on the overall Ca load of the cell. Furthermore, we show that the stochastic nature of the ryanodine receptor channel is crucial to the wave generation process.

Synthetic Lipid Nanotubes as Cell-Cell Junctions for Inter-Cellular Ca+ Propagation and for Cell Contraction Monitoring

We previously discovered that the main component of bacterial cell membranes, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), self-assembles into single-wall tubes with an outer diameter of 19.1 ± 4.5 nm and a length of close to 1 mm (synthetic lipid nanotubes, LNTs). 1By exploiting the interaction of living cells (REF52) with LNTs, we demonstrated cell contractility recording. The newly generated LNTs pattern upon cell spreading exhibited structural fingerprints characteristic for cell types and conditions. Monitoring the fluorescent-labeled LNTs and paxillin accumulated at the focal adhesions of fibroblasts simultaneously, we proved that the nucleation of new LNTs is linked to the cell's focal adhesions and contractile activity.Tunneling nanotubes (TNTs) are tube-shaped cell membrane structures between cells.2 The characterization of TNTs' functionality by traditional biological approaches often suffers from the complexities and a lack of controllability in the connection. We developed synthetic TNTs by connecting cells with the LNTs as a model system to investigate this new cell-cell communication tool. Cells (REF52 and PC12) were rinsed with physiological buffer solution to remove associated proteins, and seeded on surface-patterned LNTs. Dye-tagged phospholipids incorporated in the LNTs diffused into the cell plasma membrane, suggesting the fusion between the LNTs and the cell membrane. Physical parameters (e.g. temperature, osmotic pressure) were optimized to promote the fusion efficiency. TNTs were found to mediate the calcium wave propagation in different cells types. It implies that the inter-cellular calcium migration does not require the direct cell-cell contact with formation of gap junctions but occurs remotely through TNTs over much longer distance. We study this TNT-mediated calcium propagation by fabricating several artificial TNT-cell circuits of different geometries.1. K. Sugihara, et al., ACS nano 6, 6626 (2012).2. A. Rustom, et al., Science 303, 1007 (2004).

2F12 引張刺激が細胞集団で惹起する細胞内Ca^<2+>上昇の開始点観察(GS1-1:細胞のバイオメカニクス(1))

2F12 引張刺激が細胞集団で惹起する細胞内Ca^<2+>上昇の開始点観察(GS1-1:細胞のバイオメカニクス(1))

Regulation of the major vacuolar Ca2+ transporter genes, by intercellular Ca2+ concentration and abiotic stresses, in tip-burn resistant Brassica oleracea

Calcium is an essential plant macronutrient that has unique structural and signaling roles related to tip-burn disorder in Brassica spp. crops. For two types of cabbage inbred lines, tip-burn susceptible and resistant, we measured and compared major macronutrient cations, including Ca(2+), in leaves. In both lines, Ca(2+), Mg(2+), Na(+), and K(+), accumulated more in leaf base than in leaf apex. Ca(2+) and K(+) were >2 times more abundant in the tip-burn resistant line, while Na(+) was higher in the susceptible line. Ca(2+) differences between the two lines resulted from differential accumulation of calcium into cell vacuoles. We profiled major vacuolar Ca(2+) transporters, in both cabbage lines, by growth time and intercellular Ca(2+) concentration. Expression pattern of several Ca(2+) transporter genes differed between tip-burn susceptible and resistant lines by growth time points. We also identified promoter regions of the major Ca(2+) vacuole transporter genes, CAX1, ACA4, and ACA11, which displayed hormonal, light and defense-related cis-acting regulatory elements. Finally, transporter genes in the two cabbage lines responded differently to abiotic stresses, demonstrating diversity in gene regulation among orthologous genes.

Coordination of Intercellular Ca2+ Signaling in Endothelial Cell Tubes of Mouse Resistance Arteries

To test the hypothesis that Ca(2+) responses to GPCR activation are coordinated between neighboring ECs of resistance arteries. EC tubes were freshly isolated from superior epigastric arteries of C57BL/6 mice. Intercellular coupling was tested using microinjection of propidium iodide. Following loading with fluo-4 dye, intracellular Ca(2+) responses to ACh were imaged with confocal microscopy. Cell-to-cell transfer of propidium iodide confirmed functional GJCs. A 1 μm ACh stimulus evoked Ca(2+) responses (9.8 ± 0.8/min, F/F(0) = 3.11 ± 0.2) which pseudo-line-scan analysis revealed as composed of Ca(2+) waves and spatially restricted Ca(2+) release events. A 100 nm ACh stimulus induced Ca(2+) responses of lower frequency (4.5 ± 0.7/min) and amplitude (F/F(0) = 1.95 ± 0.11) composed primarily of spatially restricted events. The time interval between Ca(2+) waves in adjacent cells (0.79 ± 0.12 s) was shorter (p < 0.05) than that between nonadjacent cells (1.56 ± 0.25 s). Spatially restricted Ca(2+) release events had similar frequencies and latencies between adjacent and nonadjacent cells. Inhibiting intracellular Ca(2+) release with 2-APB, Xestospongin C or thapsigargin eliminated Ca(2+) responses. With moderate GPCR stimulation, localized Ca(2+) release events predominate among cells. Greater GPCR stimulation evokes coordinated intercellular Ca(2+) waves via the ER. Calcium signaling during GPCR activation is complex among cells, varying with stimulus intensity and proximity to actively signaling cells.

Permeation of Calcium through Purified Connexin 26 Hemichannels

Indirect evidence suggests that connexin hemichannels are permeable to Ca(2+), but direct demonstration is lacking. Calcium moves into liposomes containing purified Cx26 in response to a concentration gradient. Cx26 hemichannels are permeable to Ca(2+). Cx26 hemichannels may play a role in Ca(2+) influx into cells under conditions that lead to hemichannel activation, such as ischemic damage. Gap junction channels communicate the cytoplasms of two cells and are formed by head to head association of two hemichannels, one from each of the cells. Gap junction channels and hemichannels are permeable to ions and hydrophilic molecules of up to M(r) 1,000, including second messengers and metabolites. Intercellular Ca(2+) signaling can occur by movement of a number of second messengers, including Ca(2+), through gap junction channels, or by a paracrine pathway that involves activation of purinergic receptors in neighboring cells following ATP release through hemichannels. Understanding Ca(2+) permeation through Cx26 hemichannels is important to assess the role of gap junction channels and hemichannels in health and disease. In this context, it is possible that increased Ca(2+) influx through hemichannels under ischemic conditions contributes to cell damage. Previous studies suggest Ca(2+) permeation through hemichannels, based on indirect arguments. Here, we demonstrate for the first time hemichannel permeability to Ca(2+) by measuring Ca(2+) transport through purified Cx26 hemichannels reconstituted in liposomes. We trapped the low affinity Ca(2+)-sensitive fluorescent probe Fluo-5N into the liposomes and followed the increases in intraliposomal [Ca(2+)] in response to an imposed [Ca(2+)] gradient. We show that Ca(2+) does move through Cx26 hemichannels and that the permeability of the hemichannels to Ca(2+) is high, similar to that for Na(+). We suggest that hemichannels can be a significant pathway for Ca(2+) influx into cells under conditions such as ischemia.

Analysis of intercellular communication by flexible hydrodynamic gating on a microfluidic chip

Intercellular Ca(2+) waves are propagation of Ca(2+) transients among cells that could be initiated by chemical stimulation. Current methods for analyzing intercellular Ca(2+) waves are difficult to realize localized chemical stimulations upon the target cell without interfering with adjacent contacting cells. In this paper, a simple and flexible microfluidic method was developed for investigating the intercellular communication of Ca(2+) signals. A cross-patterned microfluidic chip was designed and fabricated with polydimethylsiloxane as the structural material. Localized chemical stimulation was achieved by a new strategy based on hydrodynamic gating technique. Clusters of target cells were seeded at the location within 300 μm downstream of the intersection of the cross-shaped microchannel. Confined lateral molecular diffusion largely minimized the interference from diffusion-induced stimulation of adjacent cells. Localized stimulation of the target cell with adenosine 5'-triphosphate successfully induced the propagation of intercellular Ca(2+) waves among a population of adjacent contacting cells. Further inhibition studies verified that the propagation of calcium signals among NIH-3 T3 cells was dependent on direct cytosolic transfer via gap junctions. The developed microfluidic method provides a versatile platform for investigating the dynamics of intercellular communications.

Synthesis and application of intercellular Ca2 +-sensitive fluorescent probe based on quantum dots

A novel Ca2+-sensitive fluorescent probe was synthesized and characterized with a coupled method that coupled di[2-(N,N-dicarboxylmethyl)amino]ethyl ether (EGTA) to the surface of mercaptoethylamine-modified CdTe quantum dots (CdTe/MA-EGTA QDs). The application of this probe to detect intercellular Ca2+ change in the leaf cells of Arabidopsis thaliana was studied. Results from transmission electron micrographs showed that the particle size of CdTe/MA-EGTA was about 3–4nm; the fluorescent spectrum indicated that the excitation spectral ranged from 350 to 490nm with a narrow and symmetric emission spectral peak at 565nm when excited by 400nm, and capillary electrophoresis demonstrated that CdTe/MA-EGTA was obtained by a coupling reaction. When the detected conditions were set as an excitation wavelength of 514nm and detection wavelength of 561–604nm, the increase of Ca2+ in A. thaliana leaf cells and the rapidly quenching effect of fluorescence signal induced by exogenous treatment of jasmonate acid (JA) could be measured using laser scanning confocal microscopy. The quenching rate of traditional Ca2+-sensitive fluorescent probe Fluo-3 reached about 80% within a minute when exciting at 488nm, which was much faster than the novel fluorescent probe CdTe/MA-EGTA. CdTe/MA-EGTA, however, was better at resisting photo bleaching and was more suitable for long-term tracking and monitoring than Fluo-3.

The Intercellular Synchronization of Ca2+ Oscillations Evaluates Cx36-Dependent Coupling

Connexin36 (Cx36) plays an important role in insulin secretion by controlling the intercellular synchronization of Ca2+ transients induced during stimulation. The lack of drugs acting on Cx36 channels is a major limitation in further unraveling the molecular mechanism underlying this effect. To screen for such drugs, we have developed an assay allowing for a semi-automatic, fluorimetric quantification of Ca2+ transients in large populations of MIN6 cells. Here, we show that (1) compared to control cells, MIN6 cells with reduced Cx36 expression or function showed decreased synchrony of glucose-induced Ca2+ oscillations; (2) glibenclamide, a sulphonylurea which promotes Cx36 junctions and coupling, increased the number of synchronous MIN6 cells, whereas quinine, an antimalarial drug which inhibits Cx36-dependent coupling, decreased this proportion; (3) several drugs were identified that altered the intercellular Ca2+ synchronization, cell coupling and distribution of Cx36; (4) some of them also affected insulin content. The data indicate that the intercellular synchronization of Ca2+ oscillations provides a reliable and non-invasive measurement of Cx36-dependent coupling, which is useful to identify novel drugs affecting the function of β-cells, neurons, and neuron-related cells that express Cx36.

Role of connexin40 in the autoregulatory response of the afferent arteriole

Connexins in renal arterioles affect autoregulation of arteriolar tonus and renal blood flow and are believed to be involved in the transmission of the tubuloglomerular feedback (TGF) response across the cells of the juxtaglomerular apparatus. Connexin40 (Cx40) also plays a significant role in the regulation of renin secretion. We investigated the effect of deleting the Cx40 gene on autoregulation of afferent arteriolar diameter in response to acute changes in renal perfusion pressure. The experiments were performed using the isolated blood perfused juxtamedullary nephron preparation in kidneys obtained from wild-type or Cx40 knockout mice. Renal perfusion pressure was increased in steps from 75 to 155 mmHg, and the response in afferent arteriolar diameter was measured. Hereafter, a papillectomy was performed to inhibit TGF, and the pressure steps were repeated. Conduction of intercellular Ca(2+) changes in response to local electrical stimulation was examined in isolated interlobular arteries and afferent arterioles from wild-type or Cx40 knockout mice. Cx40 knockout mice had an impaired autoregulatory response to acute changes in renal perfusion pressure compared with wild-type mice. Inhibition of TGF by papillectomy significantly reduced autoregulation of afferent arteriolar diameter in wild-type mice. In Cx40 knockout mice, papillectomy did not affect the autoregulatory response, indicating that these mice have no functional TGF. Also, Cx40 knockout mice showed no conduction of intercellular Ca(2+) changes in response to local electrical stimulation of interlobular arteries, whereas the Ca(2+) response to norepinephrine was unaffected. These results suggest that Cx40 plays a significant role in the renal autoregulatory response of preglomerular resistance vessels.

Low extracellular Ca2+ conditions induce an increase in brain endothelial permeability that involves intercellular Ca2+ waves

The intracellular calcium concentration ([Ca(2+)](i)) is an important factor determining the permeability of endothelial barriers including the blood-brain barrier (BBB). However, nothing is known concerning the effect of spatially propagated intercellular Ca(2+) waves (ICWs). The propagation of ICWs relies in large part on channels formed by connexins that are present in endothelia. We hypothesized that ICWs may result in a strong disturbance of endothelial function, because the [Ca(2+)](i) changes are coordinated and involve multiple cells. Thus, we aimed to investigate the effect of ICWs on endothelial permeability. ICW activity was triggered in immortalized and primary brain endothelial cells by lowering the extracellular Ca(2+) concentration. Low extracellular Ca(2+) increased the endothelial permeability and this was significantly suppressed by buffering [Ca(2+)](i) with BAPTA-AM, indicating a central role of [Ca(2+)](i) changes. The endothelial permeability increase was furthermore inhibited by the connexin channel blocking peptide Gap27, which also blocked the ICWs, and by inhibiting protein kinase C (PKC), Ca(2+)/calmodulin-dependent kinase II (CaMKII) and actomyosin contraction. We compared these observations with the [Ca(2+)](i) changes and permeability alterations provoked by the inflammatory agent bradykinin (BK), which triggers oscillatory [Ca(2+)](i) changes without wave activity. BK-associated [Ca(2+)](i) changes and the endothelial permeability increase were significantly smaller than those associated with ICWs, and the permeability increase was not influenced by inhibition of PKC, CaMKII or actomyosin contraction. We conclude that ICWs significantly increase endothelial permeability and therefore, the connexins that underlie wave propagation form an interesting target to limit BBB alterations. This article is part of a Special Issue entitled Electrical Synapses.

Criticality in Intracellular Calcium Signaling in Cardiac Myocytes

Calcium (Ca) is a ubiquitous second messenger that regulates many biological functions. The elementary events of local Ca signaling are Ca sparks, which occur randomly in time and space, and integrate to produce global signaling events such as intra- and intercellular Ca waves and whole-cell Ca oscillations. Despite extensive experimental characterization in many systems, the transition from local random to global synchronous events is still poorly understood. Here we show that criticality, a ubiquitous dynamical phenomenon in nature, is responsible for the transition from local to global Ca signaling. We demonstrate this first in a computational model of Ca signaling in a cardiac myocyte and then experimentally in mouse ventricular myocytes, complemented by a theoretical agent-based model to delineate the underlying dynamics. We show that the interaction between the Ca release units via Ca-induced Ca release causes self-organization of Ca spark clusters. When the coupling between Ca release units is weak, the cluster-size distribution is exponential. As the interactions become strong, the cluster-size distribution changes to a power-law distribution, which is characteristic of criticality in thermodynamic and complex nonlinear systems, and facilitates the formation and propagation of Ca waves and whole-cell Ca oscillations. Our findings illustrate how criticality is harnessed by a biological cell to regulate Ca signaling via self-organization of random subcellular events into cellular-scale oscillations, and provide a general theoretical framework for the transition from local Ca signaling to global Ca signaling in biological cells.

Long-Range Ca2+ Waves Transmit Brain-Damage Signals to Microglia

Microglia are the resident phagocytes of the brain that are responsible for the clearance of injured neurons, an essential step in subsequent tissue regeneration. How death signals are controlled both in space and time to attract these cells toward the site of injury is a topic of great interest. To this aim, we have used the optically transparent zebrafish larval brain and identified rapidly propagating Ca2+ waves that determine the range of microglial responses to neuronal cell death. We show that while Ca2+-mediated microglial responses require ATP, the spreading of intercellular Ca2+ waves is ATP independent. Finally, we identify glutamate as a potent inducer of Ca2+-transmitted microglial attraction. Thus, this real-time analysis reveals the existence of a mechanism controlling microglial targeted migration to neuronal injuries that is initiated by glutamate and proceeds across the brain in the form of a Ca2+ wave.

Abstract 3172: Role of miRNA-211in melanocytes and melanoma

Abstract MicroRNAs (miRNAs) are small non-coding regulatory RNAs that regulate gene expression. Evidence is accumulating that miRNAs are involved in almost all aspects of tumor biology including tumor cell survival, proliferation and migration. Here, we report that miRNA-211, which maps to the sixth intron of TRPM1 gene (a suppressor of melanoma metastasis), is highly expressed in primary melanocytes and its expression is reduced in primary and metastatic melanoma cell lines. Expression of miRNA-211 correlates with expression of TRPM1, a transient receptor potential family member calcium channel protein that we showed to be involved in regulation of melanocyte calcium homeostasis and melanin pigmentation. Inhibition of miRNA-211 expression by anti-miR211 inhibited intercellular Ca+2 update and the growth of rapidly growing melanocytes. Ectopic expression of miRNA-211in melanoma cells inhibit growth and reduced their migration. Regulation of miRNA-211 and its function in melanocytes and melanoma have not been well understood. We show that ectopic expression of p53 (a tumor suppressor) in melanocytes increase miRNA-211 expression and decrease TRPM1 expression. Inhibition of miRNA-211 did not change TPRM1 expression in melanocytes. Additionally, we show that 1.4 kb intron 6 genomic region of TRPM1 which contain miRNA-211 region is sufficient to activate miRNA-211 expression. And 1.4 kb of 6th intron luciferase reporter plasmid transfection followed by luciferase assay showed increase in promoter driven luciferase activity. These data suggests that the presence of putative promoter region of miRNA-211 in the sixth intron of TRPM1 gene. We propose that activation of endogenous miRNA-211 expression or targeted delivery of miR-211 in melanoma is a potential therapeutic option for treatment of cutaneous melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3172. doi:1538-7445.AM2012-3172