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

From free to effective diffusion coefficients in fluorescence correlation spectroscopy experiments

Diffusion is one of the main transport processes that occur inside cells determining the spatial and time distribution of relevant action molecules. In most cases these molecules not only diffuse but also interact with others as they get transported. When these interactions occur faster than diffusion the resulting transport can be characterized by “effective diffusion coefficients” that depend on both the reaction rates and the “free” diffusion coefficients. Fluorescence correlation spectroscopy (FCS) gives information on effective rather than free diffusion coefficients under this condition. In the present paper we investigate what coefficients can be drawn from FCS experiments for a wide range of values of the ratio of reaction to diffusion time scales, using different fitting functions. We find that the effective coefficients can be inferred with relatively small errors even when the condition of fast reactions does not exactly hold. Since the diffusion time scale depends on the size of the observation volume and the reaction time scale depends on concentrations, we also discuss how by changing either one or the other property one can switch between the two limits and extract more information on the system under study.Fil: Perez Ipiña, Emiliano. 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; ArgentinaFil: Ponce Dawson, Silvina Martha. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentin

The effect of reactions on the formation and readout of the gradient of Bicoid

During early development, the establishment of gradients of transcriptional factors determinesthe patterning of cell fates. The case of Bicoid (Bcd) in Drosophila melanogaster embryos is welldocumented and studied. There are still controversies as to whether SDD models in which Bcd isSynthesized at one end, then Diffuses and is Degraded can explain the gradient formation withinthe timescale observed experimentally. The Bcd gradient is observed in embryos that express aBicoid-eGFP fusion protein (Bcd-GFP) which cannot differentiate if Bcd is freely diffusing orbound to immobile sites. In this work we analyze an SDID model that includes the Interaction ofBcd with binding sites. We simulate numerically the resulting full reaction?diffusion system in acylindrical domain using previously determined biophysical parameters and a simplified version ofthe Bcd source. In this way we obtain solutions that depend on the spatial location approximately asobserved experimentally and that reach such dependence at a time that is also compatible with theexperimental observations. Analyzing the differences between the free and bound Bcd distributionswe observe that the latter spans over a longer lengthscale. We conclude that deriving the lengthscalefrom the distribution of Bcd-GFP can lead to an overestimation of the gradient lengthscale and ofthe Hill coefficient that relates the concentrations of Bcd and of the protein, Hunchback, whoseproduction is regulated by Bcd.Fil: Perez Ipiña, Emiliano. Universite Nice; Francia. 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; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; 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

How long should a system be observed to obtain reliable concentration estimates from the measurement of fluctuations?

The interior of cells is a highly fluctuating environment. Fluctuations set limits to the accuracy with which endogenous processes can occur. The physical principles that rule these limits also affect the experimental quantification of biophysical parameters in situ. The characterization of fluctuations, on the other hand, provides a way to quantify biophysical parameters. But as with any random process, enough data has to be collected to achieve a reliable quantitative description. In this article we study the accuracy with which intracellular concentrations can be estimated using fluorescence correlation spectroscopy. We show that, when the observed molecules interact with immobile species or experience other restrictions to their movement, the hypotheses commonly used to estimate concentrations are no longer valid. The interactions with immobile sites reduce the fluorescence variance by a finite amount. The time that is necessary to obtain an accurate concentration estimate, on the other hand, is hundreds of times larger than the slowest correlation time and is much larger when the sites move slowly than when they are immobile. Our analysis is applicable to other related techniques and it also sheds light on the way in which effector concentrations are read by target molecules in cells.Fil: Perez Ipiña, Emiliano. 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. 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; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentin

Fluctuations, correlations and the estimation of concentrations inside cells

Information transmission in cells occurs quite accurately even when concentration changes are "read" by individual binding sites. In this paper we study ligand number and site occupancy fluctuations when ligands diffuse and react going beyond the analyses that focus on their asymptotic decay. In this way we show that, for immobile binding sites, fluctuations in the number of bound molecules decay on a relatively fast scale before the asymptotic behavior kicks in. This result can explain the observed co-existence of highly fluctuating instantaneous transcriptional activities with accumulated mRNA concentrations that have relatively small noise levels. We also show that the initial stages of the decay in the bound molecule number fluctuations have one or two characteristic timescales depending on the concentration of free molecules. This transition can explain the changes in enzyme activity observed at the single molecule level.Fil: 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; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; 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

Fluorescence fluctuations and equivalence classes of Ca²⁺ imaging experiments.

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 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 IP₃Rs. 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

Statistics of pathogenic bacteria in the search of host cells

International audienceAbstract A crucial phase in the infection process, which remains poorly understood, is the localization of suitable host cells by bacteria. It is often assumed that chemotaxis plays a key role during this phase. Here, we report a quantitative study on how Salmonella Typhimurium search for T84 human colonic epithelial cells. Combining time-lapse microscopy and mathematical modeling, we show that bacteria can be described as chiral active particles with strong active speed fluctuations, which are of biological, as opposed to thermal, origin. We observe that there exists a giant range of inter-individual variability of the bacterial exploring capacity. Furthermore, we find Salmonella Typhimurium does not exhibit biased motion towards the cells and show that the search time statistics is consistent with a random search strategy. Our results indicate that in vitro localization of host cells, and also cell infection, are random processes, not involving chemotaxis, that strongly depend on bacterial motility parameters

Fluorescence fluctuations obtained from Type I–II experiments performed in oocytes with the set of concentrations (<i>ii</i>).

<p>Similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095860#pone-0095860-g003" target="_blank">Fig. 3</a> but for set (<b><i>ii</i></b>). The experimental data and their corresponding fits are shown for: (<b>A</b>) 84 images obtained in Type I experiments, fit: ; (<b>B</b>) 21 images obtained in Type II experiments, fit: . In this case the results derived from Type III experiments are not shown because no change in fluorescence was observed upon microinjection for this set of concentrations.</p