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Fluorescence fluctuations and equivalence classes of Ca²? imaging experiments.

ABSTRACT: Ca²? release into the cytosol through inositol 1,4,5-trisphosphate receptors (IP?Rs) plays a relevant role in numerous physiological processes. IP?R-mediated Ca²? signals involve Ca²?-induced Ca²?-release (CICR) whereby Ca²? release through one open IP?R induces the opening of other channels. IP?Rs are apparently organized in clusters. The signals can remain localized (i.e., Ca²? puffs) if CICR is limited to one cluster or become waves that propagate between clusters. Ca²? puffs are the building blocks of Ca²? waves. Thus, there is great interest in determining puff properties, especially in view of the current controversy on the spatial distribution of activatable IP?Rs. Ca²? puffs have been observed in intact cells with optical techniques proving that they are intrinsically Ca²? dyes, slow exogenous buffers (e.g., EGTA) to disrupt inter-cluster CICR and UV-photolyzable caged IP3. Single-wavelength dyes increase their fluorescence upon calcium binding producing images that are strongly dependent on their kinetic, transport and photophysical properties. Determining the artifacts that the imaging setting introduces is particularly relevant when trying to analyze the smallest Ca²? signals. In this paper we introduce a method to estimate the expected signal-to-noise ratio of Ca²? imaging experiments that use single-wavelength dyes. The method is based on the Number and rightness technique. It involves the performance of a series of experiments and their subsequent analysis in terms of a fluorescence fluctuation model with which the model parameters are quantified. Using the model, the expected signal-to-noise ratio is then computed. Equivalence classes between different experimental conditions that produce images with similar signal-to-noise ratios can then be established. The method may also be used to estimate the smallest signals that can reliably be observed with each setting.

SUBMITTER: Piegari E

PROVIDER: S-EPMC4002475 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Fluorescence fluctuations and equivalence classes of Ca²⁺ imaging experiments.

Piegari Estefanía E Lopez Lucía L Perez Ipiña Emiliano E Ponce Dawson Silvina S

PloS one 20140428 4

Ca²⁺ release into the cytosol through inositol 1,4,5-trisphosphate receptors (IP₃Rs) plays a relevant role in numerous physiological processes. IP₃R-mediated Ca²⁺ signals involve Ca²⁺-induced Ca²⁺-release (CICR) whereby Ca²⁺ release through one open IP₃R induces the opening of other channels. IP₃Rs are apparently organized in clusters. The signals can remain localized (i.e., Ca²⁺ puffs) if CICR is limited to one cluster or become waves that propagate between clusters. Ca²⁺ puffs are the building ...[more]

PMID: 24776736

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Project description:Intracellular calcium (Ca(2+)) plays a significant role in many cell signaling pathways, some of which are localized to spatially restricted microdomains. Ca(2+) binding proteins (Ca(2+) buffers) play an important role in regulating Ca(2+) concentration ([Ca(2+)]). Buffers typically slow [Ca(2+)] temporal dynamics and increase the effective volume of Ca(2+) domains. Because fluctuations in [Ca(2+)] decrease in proportion to the square-root of a domain's physical volume, one might conjecture that buffers decrease [Ca(2+)] fluctuations and, consequently, mitigate the significance of small domain volume concerning Ca(2+) signaling. We test this hypothesis through mathematical and computational analysis of idealized buffer-containing domains and their stochastic dynamics during free Ca(2+) influx with passive exchange of both Ca(2+) and buffer with bulk concentrations. We derive Langevin equations for the fluctuating dynamics of Ca(2+) and buffer and use these stochastic differential equations to determine the magnitude of [Ca(2+)] fluctuations for different buffer parameters (e.g., dissociation constant and concentration). In marked contrast to expectations based on a naive application of the principle of effective volume as employed in deterministic models of Ca(2+) signaling, we find that mobile and rapid buffers typically increase the magnitude of domain [Ca(2+)] fluctuations during periods of Ca(2+) influx, whereas stationary (immobile) Ca(2+) buffers do not. Also contrary to expectations, we find that in the absence of Ca(2+) influx, buffers influence the temporal characteristics, but not the magnitude, of [Ca(2+)] fluctuations. We derive an analytical formula describing the influence of rapid Ca(2+) buffers on [Ca(2+)] fluctuations and, importantly, identify the stochastic analog of (deterministic) effective domain volume. Our results demonstrate that Ca(2+) buffers alter the dynamics of [Ca(2+)] fluctuations in a nonintuitive manner. The finding that Ca(2+) buffers do not suppress intrinsic domain [Ca(2+)] fluctuations raises the intriguing question of whether or not [Ca(2+)] fluctuations are a physiologically significant aspect of local Ca(2+) signaling.

Dataset Information

Fluorescence fluctuations and equivalence classes of Ca²? imaging experiments.

Publications

Fluorescence fluctuations and equivalence classes of Ca²⁺ imaging experiments.

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