Fluorescence Fluctuations and Equivalence Classes of Ca 2+ Imaging Experiments

release into the cytosol through inositol 1,4,5-trisphosphate receptors (IP₃Rs) plays a relevant role in numerous physiological processes. IP₃R-mediated signals involve -induced -release (CICR) whereby release through one open IP₃R induces the opening of other channels. IP₃Rs are apparently organized in clusters. The signals can remain localized (i.e., puffs) if CICR is limited to one cluster or become waves that propagate between clusters. puffs are the building blocks of waves. Thus, there is great interest in determining puff properties, especially in view of the current controversy on the spatial distribution of activatable IP₃Rs. puffs have been observed in intact cells with optical techniques proving that they are intrinsically stochastic. Obtaining a correct picture of their dynamics then entails being able to detect the whole range of puff sizes. puffs are observed using visible single-wavelength 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 signals. In this paper we introduce a method to estimate the expected signal-to-noise ratio of imaging experiments that use single-wavelength dyes. The method is based on the Number and Brightness 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.     

Combining Voltage and Calcium Imaging from Neuronal Dendrites

The ability to monitor membrane potential (V _m) and calcium (Ca²⁺) transients at multiple locations on the same neuron can facilitate further progress in our understanding of neuronal function. Here we describe a method to combine V _m and Ca²⁺ imaging using styryl voltage sensitive dyes and Fura type UV-excitable Ca²⁺ indicators. In all cases V _m optical signals are linear with membrane potential changes, but the calibration of optical signals on an absolute scale is presently possible only in some neurons. The interpretation of Ca²⁺ optical signals depends on the indicator Ca²⁺ buffering capacity relative to the cell endogenous buffering capacity. In hippocampal CA1 pyramidal neurons, loaded with JPW-3028 and 300 μM Bis-Fura-2, V _m optical signals cannot be calibrated and the physiological Ca²⁺ dynamics are compromised by the presence of the indicator. Nevertheless, at each individual site, relative changes in V _m and Ca²⁺ fluorescence signals under different conditions can provide meaningful new information on local dendritic integration. In cerebellar Purkinje neurons, loaded with JPW-1114 and 1 mM Fura-FF, V _m optical signals can be calibrated in terms of mV and Ca²⁺ optical signals quantitatively reveal the physiological changes in free Ca²⁺. Using these two examples, the method is explained in detail.     

Recent developments in isotope-aided NMR methods for supramolecular protein complexes –SAIL aromatic TROSY

BackgroundThe structure-function relationships for large protein complexes at the atomic level would be comprehensively understood, if hitherto unexplored aromatic ring NMR signals became accessible in addition to the currently used backbone amide and side-chain methyl signals.MethodsThe 82 kDa malate synthase G (MSG) proteins, selectively labeled with Trp and Phe bearing relaxation optimized isotope-labeled rings, were prepared to investigate the optimal conditions for obtaining the aromatic TROSY spectra.ResultsThe MSG proteins, selectively labeled with either [δ₁,ε₁,ε₃,η₂]-SAIL Trp or ζ-SAIL Phe, provided well-separated, narrow TROSY signals for the 12 Trp and 19 Phe residues in MSG. The signals were assigned sequence-specifically, using the set of single amino acid substitution mutants. The site-specific substitution of each Phe with Tyr or Leu induced substantial chemical shifts for the other aromatic ring signals, allowing us to identify the aromatic clusters in MSG, which were comparable to the structural domains proposed previously.ConclusionsWe demonstrated that the aromatic ring ¹³CH pairs without directly bonded ¹³C and adjacent ¹H spins provide surprisingly narrow TROSY signals, if the rings are surrounded by fully deuterated amino acids. The observed signals can be readily assigned by either the single amino acid substitution or the NOEs between the aromatic and methyl protons, if the methyl assignments are available.General significanceThe method described here should be generally applicable for difficult targets, such as proteins in lipid bilayers or possibly in living cells, thus providing unprecedented opportunities to use these new probes in structural biology.     

Direct quantification of the four individual S states in Photosystem II using EPR spectroscopy

EPR spectroscopy is very useful in studies of the oxygen evolving cycle in Photosystem II and EPR signals from the CaMn₄ cluster are known in all S states except S₄. Many signals are insufficiently understood and the S₀, S₁, and S₃ states have not yet been quantifiable through their EPR signals. Recently, split EPR signals, induced by illumination at liquid helium temperatures, have been reported in the S₀, S₁, and S₃ states. These split signals provide new spectral probes to the S state chemistry. We have studied the flash power dependence of the S state turnover in Photosystem II membranes by monitoring the split S₀, split S₁, split S₃ and S₂ state multiline EPR signals. We demonstrate that quantification of the S₁, S₃ and S₀ states, using the split EPR signals, is indeed possible in samples with mixed S state composition. The amplitudes of all three split EPR signals are linearly correlated to the concentration of the respective S state. We also show that the S₁ → S₂ transition proceeds without misses following a saturating flash at 1 °C, whilst substantial misses occur in the S₂ → S₃ transition following the second flash.     

Tracer diffusion coefficients of oxyhemoglobin a and oxyhemoglobin s in blood cells as determined by pulsed field gradient NMR

It is demonstrated that tracer diffusion coefficients can be determined for oxyhemoglobin A (HbA-O₂) and oxyhemoglobin S (HbS-O₂) in intact blood cells by means of pulsed field gradient NMR (PFG-NMR). This is possible because the method discriminates between both rapidly moving water molecules and molecules having small proton transverse relaxation times (T₂). The results indicate that only hemoglobin molecules contribute to the echo signals when large field gradients are used. The dependence of the measured diffusion coefficients on osmolarity and pH are attributed to changes in hemoglobin concentration resulting from changes in cell volume.     

Comparing Apples and Oranges: Fold-Change Detection of Multiple Simultaneous Inputs

Sensory systems often detect multiple types of inputs. For example, a receptor in a cell-signaling system often binds multiple kinds of ligands, and sensory neurons can respond to different types of stimuli. How do sensory systems compare these different kinds of signals? Here, we consider this question in a class of sensory systems – including bacterial chemotaxis- which have a property known as fold-change detection: their output dynamics, including amplitude and response time, depends only on the relative changes in signal, rather than absolute changes, over a range of several decades of signal. We analyze how fold-change detection systems respond to multiple signals, using mathematical models. Suppose that a step of fold F₁ is made in input 1, together with a step of F₂ in input 2. What total response does the system provide? We show that when both input signals impact the same receptor with equal number of binding sites, the integrated response is multiplicative: the response dynamics depend only on the product of the two fold changes, F₁F₂. When the inputs bind the same receptor with different number of sites n₁ and n₂, the dynamics depend on a product of power laws, . Thus, two input signals which vary over time in an inverse way can lead to no response. When the two inputs affect two different receptors, other types of integration may be found and generally the system is not constrained to respond according to the product of the fold-change of each signal. These predictions can be readily tested experimentally, by providing cells with two simultaneously varying input signals. The present study suggests how cells can compare apples and oranges, namely by comparing each to its own background level, and then multiplying these two fold-changes.     

The Role of Photon Scattering in Voltage-Calcium Fluorescent Recordings of Ventricular Fibrillation

Recent optical mapping studies of cardiac tissue suggest that membrane voltage (V_m) and intracellular calcium concentrations (Ca) become dissociated during ventricular fibrillation (VF), generating a proarrhythmic substrate. However, experimental methods used in these studies may accentuate measured dissociation due to differences in fluorescent emission wavelengths of optical voltage/calcium (V_opt/Ca_opt) signals. Here, we simulate dual voltage-calcium optical mapping experiments using a monodomain-Luo-Rudy ventricular-tissue model coupled to a photon-diffusion model. Dissociation of both electrical, V_m/Ca, and optical, V_opt/Ca_opt, signals is quantified by calculating mutual information (MI) for VF and rapid pacing protocols. We find that photon scattering decreases MI of V_opt/Ca_opt signals by 23% compared to unscattered V_m/Ca signals during VF. Scattering effects are amplified by increasing wavelength separation between fluorescent voltage/calcium signals and respective measurement-location misalignment. In contrast, photon scattering does not affect MI during rapid pacing, but high calcium dye affinity can decrease MI by attenuating alternans in Ca_opt but not in V_opt. We conclude that some dissociation exists between voltage and calcium at the cellular level during VF, but MI differences are amplified by current optical mapping methods.     

Repeat sequences in the Bordetella pertussis adenylate cyclase toxin can be recognized as alternative carboxy-proximal secretion signals by the Escherichia coliα-haemolysin translocator

The 1706-residue adenylate cyclase toxin (CyaA) of Bordetella pertussis is an RTX protein with extensive carboxy-proximai glycine and aspartate-rich repeats. CyaA does not have a cleavable amino-terminal signal peptide and can be secreted across both bacterial membranes of the Escherichia coli cell envelope by the α-haemolysin (HlyA) translocator (HlyBD/TolC). We performed deletion mapping of secretion signals recognized in CyaA by this heterologous translocator. Truncated proteins with N–terminal and internal deletions were secreted at levels up to 10 times higher than intact CyaA and similar to HlyA. A secretion signal recognized by HlyBD/ToiC was found within the last 74 residues of CyaA. However, secretion of CyaA was reduced but not abolished upon deletion of the last 75 or 217 residues, indicating that at least two additional secretion signals recognized by HlyBD/TolC are within CyaA. One of them was localized to the repeat sequence between residues Asp-1587 to lle-1631. Interestingly, a conserved acidic' motif (Glu/Asp)-(X)₁₁-Asp-(X)_3/5-(Glu/Asp)-(X)₁₄-Asp was found in the C-terminal sequences of HlyA, CyaA and the two secreted CyaA derivatives. We speculate that the presence and spacing of acidic residues may be an important feature of secretion signals recognized by the haemolysin translocator.     

Regulation of phosphate starvation responses in higher plants

Background

Phosphorus (P) is often a limiting mineral nutrient for plant growth. Many soils worldwide are deficient in soluble inorganic phosphate (P_i), the form of P most readily absorbed and utilized by plants. A network of elaborate developmental and biochemical adaptations has evolved in plants to enhance P_i acquisition and avoid starvation.

Scope

Controlling the deployment of adaptations used by plants to avoid P_i starvation requires a sophisticated sensing and regulatory system that can integrate external and internal information regarding P_i availability. In this review, the current knowledge of the regulatory mechanisms that control P_i starvation responses and the local and long-distance signals that may trigger P_i starvation responses are discussed. Uncharacterized mutants that have P_i-related phenotypes and their potential to give us additional insights into regulatory pathways and P_i starvation-induced signalling are also highlighted and assessed.

Conclusions

An impressive list of factors that regulate P_i starvation responses is now available, as is a good deal of knowledge regarding the local and long-distance signals that allow a plant to sense and respond to P_i availability. However, we are only beginning to understand how these factors and signals are integrated with one another in a regulatory web able to control the range of responses demonstrated by plants grown in low P_i environments. Much more knowledge is needed in this agronomically important area before real gains can be made in improving P_i acquisition in crop plants.     

Calcium signalling in glial cells

Various electrical, mechanical, and chemical stimuli, including the influences of neurotrasmitters, neuromodulators, and hormones, trigger complex changes in [Ca²⁺] _i in all types of glial cells. Glial [Ca²⁺] _i responses are controlled by coordinated activity of several molecular cascades. The initiation of [Ca²⁺] _i signal in glial cells results from activation of either plasmalemmal, or intracellular Ca²⁺ permeable channels. The interplay of different molecular cascades enables the development of agonist-specific patterns of Ca²⁺ responses. Such agonist specificity may provide the means for intracellular and intercellular information coding. Furthermore, glial [Ca²⁺] _i signals can travel with no decrement within glial networks. These intercellular Ca²⁺ waves can be regarded as a substrate for information exchange between the glial cells. Neuronal activity can trigger [Ca²⁺] _i signals in neighboring glial cells and, moreover, there is some evidence that glial [Ca²⁺] _i waves can activate neuronal electrical and/or [Ca²⁺] _i , responses. Glial Ca²⁺ signalling can be regarded as a form of glial excitability.     

The influence of pH on the EPR and redox properties of cytochrome c oxidase in detergent solution and in phospholipid vesicles

The EPR signals of oxidized and partially reduced cytochrome oxidase have been studied at pH 6.4, 7.4, and 8.4. Isolated cytochrome oxidase in both non-ionic detergent solution and in phospholipid vesicles has been used in reductive titrations with ferrocytochrome c.The g values of the low- and high-field parts of the low-spin heme signal in oxidized cytochrome oxidase are shown to be pH dependent. In reductive titrations, low-spin heme signals at g 2.6 as well as rhombic and nearly axial high-spin heme signals are found at pH 8.4, while the only heme signals appearing at pH 6.4 are two nearly axial g 6 signals. This pH dependence is shifted in the vesicles.The g 2.6 signals formed in titrations with ferrocytochrome c at pH 8.4 correspond maximally to 0.25–0.35 heme per functional unit (aa₃) of cytochrome oxidase in detergent solution and to 0.22 heme in vesicle oxidase. The total amount of high-spin heme signals at g 6 found in partially reduced enzyme is 0.45–0.6 at pH 6.4 and 0.1–0.2 at pH 8.4. In titrations of cytochrome oxidase in detergent solution the g 1.45 and g 2 signals disappear with fewer equivalents of ferrocytochrome c added at pH 8.4 compared to pH 6.4.The results indicate that the environment of the hemes varies with the pH. One change is interpreted as cytochrome a₃ being converted from a high-spin to a low-spin form when the pH is increased. Possibly this transition is related to a change of a liganded H₂O to OH^? with a concomitant decrease of the redox potential. Oxidase in phosphatidylcholine vesicles is found to behave as if it experiences a pH, one unit lower than that of the medium.     

Deterministic chaos and the first positive Lyapunov exponent: a nonlinear analysis of the human electroencephalogram during sleep

Under selected conditions, nonlinear dynamical systems, which can be described by deterministic models, are able to generate so-called deterministic chaos. In this case the dynamics show a sensitive dependence on initial conditions, which means that different states of a system, being arbitrarily close initially, will become macroscopically separated for sufficiently long times. In this sense, the unpredictability of the EEG might be a basic phenomenon of its chaotic character. Recent investigations of the dimensionality of EEG attractors in phase space have led to the assumption that the EEG can be regarded as a deterministic process which should not be mistaken for simple noise. The calculation of dimensionality estimates the degrees of freedom of a signal. Nevertheless, it is difficult to decide from this kind of analysis whether a process is quasiperiodic or chaotic. Therefore, we performed a new analysis by calculating the first positive Lyapunov exponent L ₁ from sleep EEG data. Lyapunov exponents measure the mean exponential expansion or contraction of a flow in phase space. L ₁ is zero for periodic as well as quasiperiodic processes, but positive in the case of chaotic processes expressing the sensitive dependence on initial conditions. We calculated L ₁ for sleep EEG segments of 15 healthy men corresponding to the sleep stages I, II, III, IV, and REM (according to Rechtschaffen and Kales). Our investigations support the assumption that EEG signals are neither quasiperiodic waves nor a simple noise. Moreover, we found statistically significant differences between the values of L ₁ for different sleep stages. All together, this kind of analysis yields a useful extension of the characterization of EEG signals in terms of nonlinear dynamical system theory.     

Osmotic Stress Reduces Ca2+ Signals through Deformation of Caveolae

Caveolae are membrane invaginations that can sequester various signaling proteins. Caveolae have been shown to provide mechanical strength to cells by flattening to accommodate increased volume when cells are subjected to hypo-osmotic stress. We have previously found that caveolin, the main structural component of caveolae, specifically binds Gα_q and stabilizes its activation state resulting in an enhanced Ca²⁺ signal upon activation. Here, we show that osmotic stress caused by decreasing the osmolarity in half reversibly changes the configuration of caveolae without releasing a significant portion of caveolin molecules. This change in configuration due to flattening leads to a loss in Cav1-Gα_q association. This loss in Gα_q/Cav1 association due to osmotic stress results in a significant reduction of Gα_q/phospholipase Cβ-mediated Ca²⁺ signals. This reduced Ca²⁺ response is also seen when caveolae are reduced by treatment with siRNA(Cav1) or by dissolving them by methyl-β-cyclodextran. No change in Ca²⁺ release with osmotic swelling can be seen when growth factor pathways are activated. Taken together, these results connect the mechanical deformation of caveolae to Gα_q-mediated Ca²⁺ signals.     

Differentially Timed Extracellular Signals Synchronize Pacemaker Neuron Clocks

Synchronized neuronal activity is vital for complex processes like behavior. Circadian pacemaker neurons offer an unusual opportunity to study synchrony as their molecular clocks oscillate in phase over an extended timeframe (24 h). To identify where, when, and how synchronizing signals are perceived, we first studied the minimal clock neural circuit in Drosophila larvae, manipulating either the four master pacemaker neurons (LN_vs) or two dorsal clock neurons (DN₁s). Unexpectedly, we found that the PDF Receptor (PdfR) is required in both LN_vs and DN₁s to maintain synchronized LN_v clocks. We also found that glutamate is a second synchronizing signal that is released from DN₁s and perceived in LN_vs via the metabotropic glutamate receptor (mGluRA). Because simultaneously reducing Pdfr and mGluRA expression in LN_vs severely dampened Timeless clock protein oscillations, we conclude that the master pacemaker LN_vs require extracellular signals to function normally. These two synchronizing signals are released at opposite times of day and drive cAMP oscillations in LN_vs. Finally we found that PdfR and mGluRA also help synchronize Timeless oscillations in adult s-LN_vs. We propose that differentially timed signals that drive cAMP oscillations and synchronize pacemaker neurons in circadian neural circuits will be conserved across species.     

An FTIR study on the structure of the oxygen-evolving Mn-cluster of Photosystem II in different spin forms of the S2 state

The S₂ state of the oxygen-evolving Mn-cluster of Photosystem II (PS II) is known to have different forms that exhibit the g =2 multiline and g = 4.1 EPR signals. These two spin forms are interconvertible at > 200 K and the relative amplitudes of the two signals are dependent on the species of cryoprotectant and alcohol contained in the medium. Also, it was recently found that the mutiline form can be converted to the g = 4.1 form by absorption of near-infrared light by the Mn-cluster itself at around 150 K [Boussac et al. (1996) Biochemistry 35: 6984–6989]. We have used light-induced Fourier transform infrared (FTIR) difference spectroscopy to study the structural difference in these two S₂ forms. FTIR difference spectra for S₂/S₁ as well as for S₂Q_A ^-/S₁Q_A measured at cryogenic temperatures using PS II membranes in the presence of various cryoprotectants, and monohydric alcohols did not show any specific differences except for intensities of amide I bands, which were larger when ethylene glycol or glycerol was present in addition to sucrose. This result was interpreted due to more flexible movement of the protein backbones upon S₂ formation with a higher cryoprotectant content. Light-induced difference spectra measured at 150 K using either blue light without near-infrared light or red plus near-infrared light also did not show any detectable difference. In addition, a different spectrum upon near-infrared illumination at 150 K of the PS II sample in which the S₂ state had been photogenerated at 200 K exhibited no meaningful signals. These results indicate that the two S₂ forms that give rise to the multiline and g = 4.1 signals have only minor differences, if any, in the structures of amino-acid ligands and polypeptide backbones. This conclusion suggests that conversion between the two spin states is caused by a spin-state transition in the Mn(III) ion rather than valence swapping within the Mn-cluster that would considerably affect the vibrations of ligands.This revised version was published online in October 2005 with corrections to the Cover Date.     

An efficient reduced-lattice model of IP3R for probing Ca2+ dynamics

Numerous cellular processes are regulated by Ca²⁺ signals, and the endoplasmic reticulum (ER) membrane's inositol triphosphate receptor (IP₃R) is critical for modulating intracellular Ca²⁺ dynamics. The IP₃Rs are seen to be clustered in a variety of cell types. The combination of IP₃Rs clustering and IP₃Rs-mediated Ca²⁺-induced Ca²⁺ release results in the hierarchical organization of the Ca²⁺ signals, which challenges the numerical simulation given the multiple spatial and temporal scales that must be covered. The previous methods rather ignore the spatial feature of IP₃Rs or fail to coordinate the conflicts between the real biological relevance and the computational cost. In this work, a general and efficient reduced-lattice model is presented for the simulation of IP₃Rs-mediated multiscale Ca²⁺ dynamics. The model highlights biological details that make up the majority of the calcium events, including IP₃Rs clustering and calcium domains, and it reduces the complexity by approximating the minor details. The model's extensibility provides fresh insights into the function of IP₃Rs in producing global Ca²⁺ events and supports the research under more physiological circumstances. Our work contributes to a novel toolkit for modeling multiscale Ca²⁺ dynamics and advances knowledge of Ca²⁺ signals.