Functional Ca2+ Channels between Channel Clusters are Necessary for the Propagation of IP3R-Mediated Ca2+ Waves

The specificity and universality of intracellular Ca2+ signals rely on the variety of spatio-temporal patterns that the Ca2+ concentration can display. Ca2+ release into the cytosol through inositol 1,4,5-trisphosphate receptors (IP 3 Rs) is key for this variety. The opening probability of IP3Rs depends on the cytosolic Ca2+ concentration. All of the dynamics are then well described by an excitable system in which the signal propagation depends on the ability of the Ca2+ released through one IP3R to induce the opening of other IP3Rs. In most cell types, IP3Rs are organized in clusters, i.e., the cytosol is a "patchy" excitable system in which the signals can remain localized (i.e., involving the release through one or more IP3Rs in a cluster), or become global depending on the efficiency of the Ca2+ -mediated coupling between clusters. The spatial range over which the signals propagate determines the responses that the cell eventually produces. This points to the importance of understanding the mechanisms that make the propagation possible. Our previous qualitative comparison between experiments and numerical simulations seemed to indicate that Ca2+ release not only occurs within the close vicinity of the clearly identifiable release sites (IP3R clusters) but that there are also functional IP3Rs in between them. In this paper, we present a quantitative comparison between experiments and models that corroborate this preliminary conclusion. This result has implications on how the Ca2+-mediated coupling between clusters works and how it can eventually be disrupted by the different Ca2+ trapping mechanisms.Fil: Piegari, Estefanía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Ponce Dawson, Silvina Martha. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentin

Sexual pheromone modulates the frequency of cytosolic Ca2+ bursts in Saccharomyces cerevisiae

Transient and highly regulated elevations of cytosolic Ca2+ control a variety of cellular processes. Bulk measurements using radioactive Ca2+ and the luminescent sensor aequorin have shown that in response to pheromone, budding yeast cells experience a rise of cytosolic Ca2+ that is mediated by two import systems composed by the Mid1-Cch1-Ecm7 protein complex, and the Fig 1 protein. Although this response has been largely studied, there is no report on Ca2+ dynamics at the single cell level. Here, using protein calcium indicators we show that both vegetative and pheromone-treated yeast cells exhibit discrete and asynchronous Ca2+ bursts. Most bursts reach maximal amplitude in 1-10 secs, span between 7 and 30 secs and decay fitting a single exponential model. In vegetative cells bursts are scarce but preferentially occur when cells are transitioning G1 and S phase. Upon pheromone presence Ca2+ burst occurrence increases dramatically, persisting during cell growth polarization. Pheromone concentration modulates burst frequency in a mechanism that depends on Mid1, Fig 1 and a third, still unidentified, import system. We also show that the calcineurin-responsive transcription factor Crz1 experiences nuclear localization bursts during the pheromone response.Fil: Carbo, Natalia. Instituto Pasteur de Montevideo; UruguayFil: Tarkowski, Nahuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Perez Ipiña, Emiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Ponce Dawson, Silvina Martha. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; ArgentinaFil: Aguilar, Pablo Sebastián. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentin

Intra-Cluster Percolation of Calcium Signals

Calcium signals are involved in a large variety of physiological processes. Their versatility relies on the diversity of spatio-temporal behaviors that the calcium concentration can display. Calcium entry through inositol 1,4,5-trisphosphate (IP) receptors (IPR's) is a key component that participates in both local signals such as “puffs” and in global waves. IPR's are usually organized in clusters on the membrane of the endoplasmic reticulum and their spatial distribution has important effects on the resulting signal. Recent high resolution observations [1] of Ca puffs offer a window to study intra-cluster organization. The experiments give the distribution of the number of IPR's that open during each puff without much processing. Here we present a simple model with which we interpret the experimental distribution in terms of two stochastic processes: IP binding and unbinding and Ca-mediated inter-channel coupling. Depending on the parameters of the system, the distribution may be dominated by one or the other process. The transition between both extreme cases is similar to a percolation process. We show how, from an analysis of the experimental distribution, information can be obtained on the relative weight of the two processes. The largest distance over which Ca-mediated coupling acts and the density of IP-bound IPR's of the cluster can also be estimated. The approach allows us to infer properties of the interactions among the channels of the cluster from statistical information on their emergent collective behavior

Quantification of fluctuations from fluorescence correlation spectroscopy experiments in reaction-diffusion systems

Fluorescence correlation spectroscopy (FCS) is commonly used to estimate diffusion and reaction rates. In FCS the fluorescence coming from a small volume is recorded and the autocorrelation function (ACF) of the fluorescence fluctuations is computed. Scaling out the fluctuations due to the emission process, this ACF can be related to the ACF of the fluctuations in the number of observed fluorescent molecules. In this paper the ACF of the molecule number fluctuations is studied theoretically, with no approximations, for a reaction-diffusion system in which the fluorescence changes with binding and unbinding. Theoretical ACFs are usually derived assuming that fluctuations in the number of molecules of one species are instantaneously uncorrelated to those of the others and obey Poisson statistics. Under these assumptions, the ACF derived in this paper is characterized only by the diffusive timescale of the fluorescent species and its total weight is the inverse of the mean number of observed fluorescent molecules. The theory is then scrutinized in view of previous experimental results which, for a similar system, gave a different total weight and correct estimates of other diffusive timescales. The total weight mismatch is corrected by assuming that the variance of the number of fluorescent molecules depends on the variance of the particle numbers of the other species, as in the variance decomposition formula. Including the finite acquisition time in its computation, it is shown that the ACF depends on various timescales of the system and that its total weight coincides with the one obtained with the variance decomposition formula. This calculation implies that diffusion coefficients of nonobservable species can be estimated with FCS experiments performed in reaction-diffusion systems. Ways to proceed in future experiments are also discussed.Fil: Villarruel, Cecilia Liliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Ponce Dawson, Silvina Martha. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin

Mean Field Strategies Induce Unrealistic Non-Linearities in Calcium Puffs

Mean field models are often useful approximations to biological systems, but sometimes, they can yield misleading results. In this work, we compare mean field approaches with stochastic models of intracellular calcium release. In particular, we concentrate on calcium signals generated by the concerted opening of several clustered channels (calcium puffs). To this end we simulate calcium puffs numerically and then try to reproduce features of the resulting calcium distribution using mean field models were all the channels open and close simultaneously. We show that an unrealistic non-linear relationship between the current and the number of open channels is needed to reproduce the simulated puffs. Furthermore, a single channel current which is five times smaller than the one of the stochastic simulations is also needed. Our study sheds light on the importance of the stochastic kinetics of the calcium release channel activity to estimate the release fluxes

A model of the IP3 receptor with a luminal calcium binding site: Stochastic simulations and analysis

We have constructed a stochastic model of the inositol 1,4,5-trisphosphate receptor-Ca2+ channel that is based on quantitative measurements of the channel's properties. It displays the observed dependence of the open probability of the channel with cytosolic [Ca2+] and [IP3] and gives values for the dwell times that agree with the observations. The model includes an explicit dependence of channel gating with luminal calcium. This not only explains several observations reported in the literature, but also provides a possible explanation of why the open probabilities and shapes of the bell-shaped curves reported in [Nature 351 (1991) 751] and in [Proc. Natl. Acad. Sci. U.S.A. 269 (1998) 7238] are so different. © 2003 Elsevier Ltd. All rights reserved.Fil: Fraiman Borrazás, Daniel Edmundo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Ponce Dawson, Silvina Martha. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentin

Un modelo de turbulencia de Alfven en plasmas

Se presenta un modelo de turbulencia de Alfven basado en una estadística de solitones de la ecuación no lineal derivada de Schrodinger (DNLS). Se desarrolla un código de resolución numérica de dicha ecuación y se lo prueba para distintas condiciones iniciales. Se obtiene un criterio para determinar bajo que condiciones iniciales tendrá lugar la formación de solitones, que resulta una versión integrada de uno obtenido previamente en la literatura (Mjolhus, 1976) para distinguir entre casos modulacionalmente estables e inestables. Se encuentra una nueva constante de movimiento para la ecuación DNLS que permite escribirla como una ecuación de Hamilton en forma canónica y se demuestra la relación existente entre las simétricas de la ecuación y el análisis de estabilidad de los solitones. El trabajo numérico sugiere la estabilidad de forma de los mismos. Se elabora un método que permite calcular, dada la condición inicial, el tren de solitones en que decaerá dicha condición al evolucionar de acuerdo a la ecuación DNLS. Se obtiene una expresión analítica para el numero de solitones y otra para la distribución de uno de los dos parámetros que definen cada soliton como funcional de las condiciones iniciales. Ambas concuerdan muy bien con resultados analíticos y números previos. Se comparan dos tratamientos de la estadística de solitones con observaciones del viento solar y se obtienen en ambos casos resultados cualitativamente correctos. Se discute luego cual es el mejor ajusta las observaciones.Fil:Ponce Dawson, Silvina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina