KRAP is required for diffuse and punctate IP3-mediated Ca2+ liberation and determines the number of functional IP3R channels within clusters.

KRas-induced actin-interacting protein (KRAP) has been identified as crucial for the appropriate localization and functioning of the inositol trisphosphate receptors (IP3Rs) that mediate Ca2+ release from the endoplasmic reticulum. Here, we used siRNA knockdown of KRAP expression in HeLa and HEK293 cells to examine the roles of KRAP in the generation of IP3-mediated local Ca2+ puffs and global, cell-wide Ca2+ signals. High resolution Ca2+ imaging revealed that the mean amplitude of puffs was strongly reduced by KRAP knockdown, whereas the Ca2+ flux during openings of individual IP3R channels was little affected. In both control and KRAP knockdown cells the numbers of functional channels in the clusters underlying puff sites were stochastically distributed following a Poisson relationship, but the mean number of functional channels per site was reduced by about two thirds by KRAP knockdown. We conclude that KRAP is required for activity of IP3R channels at puff sites and stochastically 'licenses' the function of individual channels on a one-to-one basis, rather than determining the functioning of the puff site as a whole. In addition to puff activity ('punctate' Ca2+ release), global, cell-wide Ca2+ signals evoked by higher levels of IP3 are further composed from a discrete 'diffuse' mode of Ca2+ release. By applying fluctuation analysis to isolate the punctate component during global Ca2+ signals, we find that KRAP knockdown suppresses to similar extents punctate and diffuse Ca2+ release in wild-type cells and in HEK293 cells exclusively expressing type 1 and type 3 IP3Rs. Thus, KRAP appears essential for the functioning of the IP3Rs involved in diffuse Ca2+ release as well as the clustered IP3Rs that generate local Ca2+ puffs.

Termination of Ca2+ puffs during IP3-evoked global Ca2+ signals.

We previously described that cell-wide cytosolic Ca2+ transients evoked by inositol trisphosphate (IP3) are generated by two modes of Ca2+ liberation from the ER; 'punctate' release via an initial flurry of transient Ca2+ puffs from local clusters of IP3 receptors, succeeded by a spatially and temporally 'diffuse' Ca2+ liberation. Those findings were derived using statistical fluctuation analysis to monitor puff activity which is otherwise masked as global Ca2+ levels rise. Here, we devised imaging approaches to resolve individual puffs during global Ca2+ elevations to better investigate the mechanisms terminating the puff flurry. We find that puffs contribute about 40% (∼90 attomoles) of the total Ca2+ liberation, largely while the global Ca2+ signal rises halfway to its peak. The major factor terminating punctate Ca2+ release is an abrupt decline in puff frequency. Although the amplitudes of large puffs fall during the flurry, the amplitudes of more numerous small puffs remain steady, so overall puff amplitudes decline only modestly (∼30%). The Ca2+ flux through individual IP3 receptor/channels does not measurably decline during the flurry, or when puff activity is depressed by pharmacological lowering of Ca2+ levels in the ER lumen, indicating that the termination of punctate release is not a simple consequence of reduced driving force for Ca2+ liberation. We propose instead that the gating of IP3 receptors at puff sites is modulated such that their openings become suppressed as the bulk [Ca2+] in the ER lumen falls during global Ca2+ signals.

ER-luminal [Ca2+] regulation of InsP3 receptor gating mediated by an ER-luminal peripheral Ca2+-binding protein.

Modulating cytoplasmic Ca2+ concentration ([Ca2+]i) by endoplasmic reticulum (ER)-localized inositol 1,4,5-trisphosphate receptor (InsP3R) Ca2+-release channels is a universal signaling pathway that regulates numerous cell-physiological processes. Whereas much is known regarding regulation of InsP3R activity by cytoplasmic ligands and processes, its regulation by ER-luminal Ca2+ concentration ([Ca2+]ER) is poorly understood and controversial. We discovered that the InsP3R is regulated by a peripheral membrane-associated ER-luminal protein that strongly inhibits the channel in the presence of high, physiological [Ca2+]ER. The widely-expressed Ca2+-binding protein annexin A1 (ANXA1) is present in the nuclear envelope lumen and, through interaction with a luminal region of the channel, can modify high-[Ca2+]ER inhibition of InsP3R activity. Genetic knockdown of ANXA1 expression enhanced global and local elementary InsP3-mediated Ca2+ signaling events. Thus, [Ca2+]ER is a major regulator of InsP3R channel activity and InsP3R-mediated [Ca2+]i signaling in cells by controlling an interaction of the channel with a peripheral membrane-associated Ca2+-binding protein, likely ANXA1.

IP3 mediated global Ca2+ signals arise through two temporally and spatially distinct modes of Ca2+ release.

The 'building-block' model of inositol trisphosphate (IP3)-mediated Ca2+ liberation posits that cell-wide cytosolic Ca2+ signals arise through coordinated activation of localized Ca2+ puffs generated by stationary clusters of IP3 receptors (IP3Rs). Here, we revise this hypothesis, applying fluctuation analysis to resolve Ca2+ signals otherwise obscured during large Ca2+ elevations. We find the rising phase of global Ca2+ signals is punctuated by a flurry of puffs, which terminate before the peak by a mechanism involving partial ER Ca2+ depletion. The continuing rise in Ca2+, and persistence of global signals even when puffs are absent, reveal a second mode of spatiotemporally diffuse Ca2+ signaling. Puffs make only small, transient contributions to global Ca2+ signals, which are sustained by diffuse release of Ca2+ through a functionally distinct process. These two modes of IP3-mediated Ca2+ liberation have important implications for downstream signaling, imparting spatial and kinetic specificity to Ca2+-dependent effector functions and Ca2+ transport.

Spatial-temporal patterning of Ca2+ signals by the subcellular distribution of IP3 and IP3 receptors.

The patterning of cytosolic Ca2+ signals in space and time underlies their ubiquitous ability to specifically regulate numerous cellular processes. Signals mediated by liberation of Ca2+ sequestered in the endoplasmic reticulum (ER) through inositol trisphosphate receptor (IP3R) channels constitute a hierarchy of events; ranging from openings of individual IP3 channels, through the concerted openings of several clustered IP3Rs to generate local Ca2+ puffs, to global Ca2+ waves and oscillations that engulf the entire cell. Here, we review recent progress in elucidating how this hierarchy is shaped by an interplay between the functional gating properties of IP3Rs and their spatial distribution within the cell. We focus in particular on the subset of IP3Rs that are organized in stationary clusters and are endowed with the ability to preferentially liberate Ca2+.

Applications of FLIKA, a Python-based image processing and analysis platform, for studying local events of cellular calcium signaling.

The patterning of cytosolic Ca2+ signals underlies their ubiquitous ability to specifically regulate numerous cellular processes. Advances in fluorescence microscopy have made it possible to image these signals with unprecedented temporal and spatial resolution. However, this is a double-edged sword, as the resulting enormous data sets necessitate development of software to automate image processing and analysis. Here, we describe Flika, an open source, graphical user interface program written in the Python environment that contains a suite of built-in image processing tools to enable intuitive visualization of image data and analysis. We illustrate the utility and power of Flika by three applications for studying cellular Ca2+ signaling: a script for measuring single-cell global Ca2+ signals; a plugin for the detection, localization and analysis of subcellular Ca2+ puffs; and a script that implements a novel approach for fluctuation analysis of transient, local Ca2+ fluorescence signals. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

All three IP3 receptor isoforms generate Ca2+ puffs that display similar characteristics.

Inositol 1,4,5-trisphosphate (IP3) evokes Ca2+ release through IP3 receptors (IP3Rs) to generate both local Ca2+ puffs arising from concerted openings of clustered IP3Rs and cell-wide Ca2+ waves. Imaging Ca2+ puffs with single-channel resolution yields information on the localization and properties of native IP3Rs in intact cells, but interpretation has been complicated because cells express varying proportions of three structurally and functionally distinct isoforms of IP3Rs. Here, we used TIRF and light-sheet microscopy to image Ca2+ puffs in HEK-293 cell lines generated by CRISPR-Cas9 technology to express exclusively IP3R type 1, 2, or 3. Photorelease of the IP3 analog i-IP3 in all three cell lines evoked puffs with largely similar mean amplitudes, temporal characteristics, and spatial extents. Moreover, the single-channel Ca2+ flux was similar among isoforms, indicating that clusters of different IP3R isoforms contain comparable numbers of active channels. Our results show that all three IP3R isoforms cluster to generate local Ca2+ puffs and, contrary to findings of divergent properties from in vitro electrophysiological studies, display similar conductances and gating kinetics in intact cells.

Comparison of Ca2+ puffs evoked by extracellular agonists and photoreleased IP3.

The inositol trisphosphate (IP3) signaling pathway evokes local Ca2+ signals (Ca2+ puffs) that arise from the concerted openings of clustered IP3 receptor/channels in the ER membrane. Physiological activation is triggered by binding of agonists to G-protein-coupled receptors (GPCRs) on the cell surface, leading to cleavage of phosphatidyl inositol bisphosphate and release of IP3 into the cytosol. Photorelease of IP3 from a caged precursor provides a convenient and widely employed means to study the final stage of IP3-mediated Ca2+ liberation, bypassing upstream signaling events to enable more precise control of the timing and relative concentration of cytosolic IP3. Here, we address whether Ca2+ puffs evoked by photoreleased IP3 fully replicate those arising from physiological agonist stimulation. We imaged puffs in individual SH-SY5Y neuroblastoma cells that were sequentially stimulated by picospritzing extracellular agonist (carbachol, CCH or bradykinin, BK) followed by photorelease of a poorly-metabolized IP3 analog, i-IP3. The centroid localizations of fluorescence signals during puffs evoked in the same cells by agonists and photorelease substantially overlapped (within ∼1µm), suggesting that IP3 from both sources accesses the same, or closely co-localized clusters of IP3Rs. Moreover, the time course and spatial spread of puffs evoked by agonists and photorelease matched closely. Because photolysis generates IP3 uniformly throughout the cytoplasm, our results imply that IP3 generated in SH-SY5Y cells by activation of receptors to CCH and BK also exerts broadly distributed actions, rather than specifically activating a subpopulation of IP3Rs that are scaffolded in close proximity to cell surface receptors to form a signaling nanodomain.

Communication of Ca(2+) signals via tunneling membrane nanotubes is mediated by transmission of inositol trisphosphate through gap junctions.

Tunneling membrane nanotubes (TNTs) are thin membrane projections linking cell bodies separated by many micrometers, which are proposed to mediate signaling and even transfer of cytosolic contents between distant cells. Several reports describe propagation of Ca(2+) signals between distant cells via TNTs, but the underlying mechanisms remain poorly understood. Utilizing a HeLa M-Sec cell line engineered to upregulate TNTs we replicated previous findings that mechanical stimulation elicits robust cytosolic Ca(2+) elevations that propagate to surrounding, physically separate cells. However, whereas this was previously interpreted to involve intercellular communication through TNTs, we found that Ca(2+) signal propagation was abolished - even in TNT-connected cells - after blocking ATP-mediated paracrine signaling with a cocktail of extracellular inhibitors. To then establish whether gap junctions may enable cell-cell signaling via TNTs under these conditions, we expressed sfGFP-tagged connexin-43 (Cx43) in HeLa M-Sec cells. We observed robust communication of mechanically-evoked Ca(2+) signals between distant but TNT-connected cells, but only when both cells expressed Cx43. Moreover, we also observed communication of Ca(2+) signals evoked in one cell by local photorelease of inositol 1,4,5-trisphosphate (IP3). Ca(2+) responses in connected cells began after long latencies at intracellular sites several microns from the TNT connection site, implicating intercellular transfer of IP3 and subsequent IP3-mediated Ca(2+) liberation, and not Ca(2+) itself, as the mediator between TNT-connected, Cx43-expressing cells. Our results emphasize the need to control for paracrine transmission in studies of cell-cell signaling via TNTs and indicate that, in this cell line, TNTs do not establish cytosolic continuity between connected cells but rather point to the crucial importance of connexins to enable communication of cytosolic Ca(2+) signals via TNTs.

A comparison of fluorescent Ca²âº indicators for imaging local Ca²âº signals in cultured cells.

Localized subcellular changes in Ca(2+) serve as important cellular signaling elements, regulating processes as diverse as neuronal excitability and gene expression. Studies of cellular Ca(2+) signaling have been greatly facilitated by the availability of fluorescent Ca(2+) indicators. The respective merits of different indicators to monitor bulk changes in cellular Ca(2+) levels have been widely evaluated, but a comprehensive comparison for their use in detecting and analyzing local, subcellular Ca(2+) signals is lacking. Here, we evaluated several fluorescent Ca(2+) indicators in the context of local Ca(2+) signals (puffs) evoked by inositol 1,4,5-trisphosphate (IP3) in cultured human neuroblastoma SH-SY5Y cells, using high-speed video-microscopy. Altogether, nine synthetic Ca(2+) dyes (Fluo-4, Fluo-8, Fluo-8 high affinity, Fluo-8 low affinity, Oregon Green BAPTA-1, Cal-520, Rhod-4, Asante Calcium Red, and X-Rhod-1) and three genetically-encoded Ca(2+)-indicators (GCaMP6-slow, -medium and -fast variants) were tested; criteria include the magnitude, kinetics, signal-to-noise ratio and detection efficiency of local Ca(2+) puffs. Among these, we conclude that Cal-520 is the optimal indicator for detecting and faithfully tracking local events; that Rhod-4 is the red-emitting indicator of choice; and that none of the GCaMP6 variants are well suited for imaging subcellular Ca(2+) signals.

Imaging local Ca2+ signals in cultured mammalian cells.

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of >500 frames sec(-1) (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Protein S-glutathionylation enhances Ca2+-induced Ca2+ release via the IP3 receptor in cultured aortic endothelial cells.

In non-excitable cells, thiol-oxidizing agents have been shown to evoke oscillations in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) by increasing the sensitivity of the inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) to IP(3). Although thiol modification of the IP(3)R is implicated in this response, the molecular nature of the modification(s) responsible for changes in channel activity is still not well understood. Diamide is a chemical oxidant that selectively converts reduced glutathione (GSH) to its disulfide (GSSG) and promotes the formation of protein­glutathione (P-SSG) mixed disulfide, i.e. glutathionylation. In the present study, we examined the effect of diamide, and the model oxidant hydrogen peroxide (H(2)O(2)), on oscillations in [Ca(2+)](i) in fura-2-loaded bovine (BAECs) and human (HAECs) aortic endo-thelial cells using time-lapse fluorescence video microscopy. In the absence of extracellular Ca(2+), acute treatment with either diamide or H(2)O(2) increased the number of BAECs exhibiting asynchronous Ca(2+) oscillations, whereas HAECs were unexpectedly resistant. Diamide pretreatment increased the sensitivity of HAECs to histamine-stimulated Ca(2+) oscillations and BAECs to bradykinin-stimulated Ca(2+) oscillations. Moreover, in both HAECs and BAECs, diamide dramatically increased both the rate and magnitude of the thapsigargin-induced Ca(2+) transient suggesting that Ca(2+)-induced Ca(2+) release (CICR) via the IP(3)R is enhanced by glutathionylation. Similar to diamide, H(2)O(2) increased the sensitivity of HAECs to both histamine and thapsigargin. Lastly, biochemical studies showed that glutathionylation of native IP(3)R(1) is increased in cells challenged with H(2)O(2). Collectively our results reveal that thiol-oxidizing agents primarily increase the sensitivity of the IP(3)R to Ca(2+), i.e. enhanced CICR, and suggest that glutathionylation may represent a fundamental mechanism for regulating IP(3)R activity during physiological redox signalling and during pathologicalical oxidative stress.

Phosphoinositide binding differentially regulates NHE1 Na+/H+ exchanger-dependent proximal tubule cell survival.

Tubular atrophy predicts chronic kidney disease progression, and is caused by proximal tubular epithelial cellcaused by proximal tubular epithelial cell (PTC) apoptosis. The normally quiescent Na(+)/H(+) exchanger-1 (NHE1) defends against PTC apoptosis, and is regulated by PI(4,5)P(2) binding. Because of the vast array of plasma membrane lipids, we hypothesized that NHE1-mediated cell survival is dynamically regulated by multiple anionic inner leaflet phospholipids. In membrane overlay and surface plasmon resonance assays, the NHE1 C terminus bound phospholipids with low affinity and according to valence (PIP(3) > PIP(2) > PIP = PA > PS). NHE1-phosphoinositide binding was enhanced by acidic pH, and abolished by NHE1 Arg/Lys to Ala mutations within two juxtamembrane domains, consistent with electrostatic interactions. PI(4,5)P(2)-incorporated vesicles were distributed to apical and lateral PTC domains, increased NHE1-regulated Na(+)/H(+) exchange, and blunted apoptosis, whereas NHE1 activity was decreased in cells enriched with PI(3,4,5)P(3), which localized to basolateral membranes. Divergent PI(4,5)P(2) and PI(3,4,5)P(3) effects on NHE1-dependent Na(+)/H(+) exchange and apoptosis were confirmed by selective phosphoinositide sequestration with pleckstrin homology domain-containing phospholipase Cδ and Akt peptides, PI 3-kinase, and Akt inhibition in wild-type and NHE1-null PTCs. The results reveal an on-off switch model, whereby NHE1 toggles between weak interactions with PI(4,5)P(2) and PI(3,4,5)P(3). In response to apoptotic stress, NHE1 is stimulated by PI(4,5)P(2), which leads to PI 3-kinase activation, and PI(4,5)P(2) phosphorylation. The resulting PI(3,4,5)P(3) dually stimulates sustained, downstream Akt survival signaling, and dampens NHE1 activity through competitive inhibition and depletion of PI(4,5)P(2).

Effect of protein S-glutathionylation on Ca2+ homeostasis in cultured aortic endothelial cells.

Diamide is a membrane-permeable, thiol-oxidizing agent that rapidly and reversibly oxidizes glutathione to GSSG and promotes formation of protein-glutathione mixed disulfides. In the present study, the acute effect of diamide on free cytosolic Ca2+ concentration ([Ca2+]i) was examined in fura-2-loaded bovine aortic endothelial cells. At low concentrations (50, 100 µM), diamide reversibly increased spontaneous, asynchronous Ca2+ oscillations, whereas, at higher concentrations (250, 500 µM), diamide caused an immediate synchronized Ca2+ oscillation in essentially all cells of the monolayer, followed by a time-dependent rise in basal [Ca2+]i. The effects of diamide on [Ca2+]i dynamics were independent of extracellular Ca2+. Inhibition of phospholipase C by U-73122 prevented the observed changes in [Ca2+]i. Additionally, the diamide-induced oscillations, but not the rise in basal [Ca2+]i, were blocked by inhibition of the inositol-1,4,5-trisphosphate (IP3) receptor (IP3R) by 2-aminoethyl diphenyl borate. However, diamide failed to alter the plasmalemmal distribution of a green fluorescent protein-tagged phosphatidylinositol-4,5-bisphosphate binding protein, demonstrating that diamide does not activate phospholipase C. Inhibition of glutathione reductase by N,N'-bis(2-chloroethyl)-N-nitrosourea or depletion of glutathione by l-buthionine-sulfoximine enhanced the effects of diamide, which, under these conditions, could only be reversed by addition of dithiothreitol to the wash buffer. Biochemical assays showed that both the IP3R and the plasmalemmal Ca2+-ATPase pump could be reversibly glutathionylated in response to diamide. These results demonstrate that diamide promotes Ca2+ release from IP3-sensitive internal Ca2+ stores and elevates basal [Ca2+]i in the absence of extracellular Ca2+, effects that may be related to a diamide-induced glutathionylation of the IP3R and the plasmalemmal Ca2+-ATPase Ca2+ pump, respectively.

Elevation of osteopontin levels in brain tumor cells reduces burden and promotes survival through the inhibition of cell dispersal.

Osteopontin (OPN) is a pleotrophic molecule that has been associated with multiple disorders of the central nervous system (CNS). Its roles in CNS malignancy are unclear but suggest that higher levels of OPN expression correlate with increased tumor grade and increased migratory capacity of tumor cells. In this study OPN cDNA was cloned into a retroviral vector and used to infect F98 Fischer rat-derived glioma cells and U87 human-derived glioblastoma multiforme (GBM) cells in vitro. Cells expressing high levels of OPN migrated less distance than control cells in vitro. This effect was not RGD mediated, but was reversed in the presence of c-Jun N-terminal kinase (JNK) inhibitor suggesting that JNK1 is an essential component of a negative feedback loop affecting OPN activated signaling cascades. Implantation of tumor cells expressing high levels of OPN into adult Fischer rats and nude rats resulted in morphologically distinct tumors and prolonged host survival relative to controls. We propose that local produced, high level OPN expression limits the malignant character of glioma cells and that the downstream mechanisms involved represent pathways that may have therapeutic value in the treatment of human CNS malignancy.