KRAP is required for diffuse and punctate IP-mediated Ca liberation and determines the number of functional IPR 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 (IPRs) that mediate Ca 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 IP-mediated local Ca puffs and global, cell-wide Ca signals. High resolution Ca imaging revealed that the mean amplitude of puffs was strongly reduced by KRAP knockdown, whereas the Ca flux during openings of individual IPR channels was little affected.

Termination of Ca puffs during IP-evoked global Ca signals.

We previously described that cell-wide cytosolic Ca transients evoked by inositol trisphosphate (IP) are generated by two modes of Ca liberation from the ER; 'punctate' release via an initial flurry of transient Ca puffs from local clusters of IP receptors, succeeded by a spatially and temporally 'diffuse' Ca liberation. Those findings were derived using statistical fluctuation analysis to monitor puff activity which is otherwise masked as global Ca levels rise. Here, we devised imaging approaches to resolve individual puffs during global Ca elevations to better investigate the mechanisms terminating the puff flurry.

ER-luminal [Ca] regulation of InsP receptor gating mediated by an ER-luminal peripheral Ca-binding protein.

Modulating cytoplasmic Ca concentration ([Ca]) by endoplasmic reticulum (ER)-localized inositol 1,4,5-trisphosphate receptor (InsPR) Ca-release channels is a universal signaling pathway that regulates numerous cell-physiological processes. Whereas much is known regarding regulation of InsPR activity by cytoplasmic ligands and processes, its regulation by ER-luminal Ca concentration ([Ca]) is poorly understood and controversial. We discovered that the InsPR is regulated by a peripheral membrane-associated ER-luminal protein that strongly inhibits the channel in the presence of high, physiological [Ca].

IP mediated global Ca signals arise through two temporally and spatially distinct modes of Ca release.

The 'building-block' model of inositol trisphosphate (IP)-mediated Ca liberation posits that cell-wide cytosolic Ca signals arise through coordinated activation of localized Ca puffs generated by stationary clusters of IP receptors (IPRs). Here, we revise this hypothesis, applying fluctuation analysis to resolve Ca signals otherwise obscured during large Ca elevations. We find the rising phase of global Ca signals is punctuated by a flurry of puffs, which terminate before the peak by a mechanism involving partial ER Ca depletion.

Spatial-temporal patterning of Ca signals by the subcellular distribution of IP and IP receptors.

The patterning of cytosolic Ca signals in space and time underlies their ubiquitous ability to specifically regulate numerous cellular processes. Signals mediated by liberation of Ca sequestered in the endoplasmic reticulum (ER) through inositol trisphosphate receptor (IPR) channels constitute a hierarchy of events; ranging from openings of individual IP channels, through the concerted openings of several clustered IPRs to generate local Ca puffs, to global Ca 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 IPRs and their spatial distribution within the cell.

All three IP receptor isoforms generate Ca puffs that display similar characteristics.

Inositol 1,4,5-trisphosphate (IP) evokes Ca release through IP receptors (IPRs) to generate both local Ca puffs arising from concerted openings of clustered IPRs and cell-wide Ca waves. Imaging Ca puffs with single-channel resolution yields information on the localization and properties of native IPRs in intact cells, but interpretation has been complicated because cells express varying proportions of three structurally and functionally distinct isoforms of IPRs. Here, we used TIRF and light-sheet microscopy to image Ca puffs in HEK-293 cell lines generated by CRISPR-Cas9 technology to express exclusively IPR type 1, 2, or 3.

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

The patterning of cytosolic Ca 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.

Comparison of Ca puffs evoked by extracellular agonists and photoreleased IP.

The inositol trisphosphate (IP) signaling pathway evokes local Ca signals (Ca puffs) that arise from the concerted openings of clustered IP 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 IP into the cytosol. Photorelease of IP from a caged precursor provides a convenient and widely employed means to study the final stage of IP-mediated Ca liberation, bypassing upstream signaling events to enable more precise control of the timing and relative concentration of cytosolic IP.

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.

A comparison of fluorescent Ca²⁺ indicators for imaging local Ca²⁺ 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.

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).

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 protein–glutathione (P-SSG) mixed disulfide, i.

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.

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.