生物化学

Diverse and complex molecular recognitions are central elements of signal transduction cascades. The strength and nature of these interaction modes can be determined by different experimental approaches. Among those, Isothermal titration calorimetry (ITC) offers certain advantages by providing binding constants and thermodynamic parameters from titration series without a need to label or immobilize one or more interaction partners. Furthermore, second messenger homeostasis involving Ca²⁺-ions requires in particular knowledge about stoichiometries and affinities of Ca²⁺-binding to Ca²⁺-sensor proteins or Ca²⁺-dependent regulators, which can be obtained by employing ITC. We used ITC to measure these parameters for a set of neuronal Ca²⁺-sensor proteins operating in photoreceptor cells. Here, we present a step wise protocol to (a) measure Ca²⁺ interaction with the Ca²⁺-sensor guanylate cyclase-activating protein 1, (b) to design an ITC experiment and prepare samples, (c) to remove Ca²⁺ nearly completely from Ca²⁺ binding proteins without using a chelating agent like EGTA.

Determination of the relative distribution of Ca²⁺ and Mn²⁺ is an important tool for analyzing mutants showing altered levels of calcium and/or manganese transporters in the chloroplast envelope or thylakoid membrane. The method described in this protocol allows quantitative analyses of the relative distribution of calcium and manganese ions between chloroplast stroma and thylakoids using the isotopes [⁴⁵Ca] and [⁵⁴Mn] as radioactive tracers. To avoid contaminations with non chloroplastidic membrane systems, the method is designed for isolating pure and intact chloroplasts of Arabidopsis thaliana. Intact chloroplasts are isolated via Percoll gradient centrifugation. Chloroplasts are then allowed to take up [⁴⁵Ca] or [⁵⁴Mn] during a light incubation step. After incubation, chloroplasts are either kept intact or osmotically/mechanically treated to release thylakoids. The amount of incorporated [⁴⁵Ca] or [⁵⁴Mn] can be determined by liquid scintillation counting and the relative distribution calculated.

Calcium plays important roles in maintaining plant cellular structure and also acts as a key secondary messenger in intercellular signaling. Thirty years ago, methods of detecting calcium in sub-cellular level had been established (Stockwell and Hanchey, 1982; Borgers et al., 1982) and reviewed extensively (Wick and Heplerm, 1982). We had used the method of testing calcium localization in salt tolerance improved transgenic alfalfa plant (Zhang and Wang, 2015). Here, we describe the protocol of testing calcium deposition by staining with potassium pyroantimonate (PPA) in detail, which was adapted from former reports (Stockwell and Hanchey, 1982; Borgers et al., 1982). The principle of this protocol is that the Ca²⁺ can react with antimonite and from black granules, which can be observed under a transmission electron microscope. The protocol includes common micromanipulation techniques of plant tissue, observation with a transmission electron microscope and photography.

Cytoplasmic calcium ([Ca²⁺]_cyt) acts as a stimulus-induced second messenger in multiple signal transduction cascades (Allen et al., 1999). In plant cells, a dramatic and readily assayed response to stimulus is the change of stomatal aperture. Changes in [Ca²⁺]_cyt of stomatal guard cells were involved in stomatal movement in response to various stimuli and cellular processes. In general, there are two available ways to measure [Ca²⁺]_cyt in guard cells, i.e., loading of calcium-sensitive fluorescence dyes such as fluo-3 AM and fura-2 or expressing genetically encoded calcium indicators such as yellow cameleon (Krebs et al., 2012). In this protocol, we aim at describing the experimental procedure to record [Ca²⁺]_cyt fluctuation in guard cells with loading of fluo-3 AM upon ABA or PA treatment combining with fluorescence imaging performed with confocal laser scanning microscope.

Biomineralization in vertebrates has both physiological and pathological aspects. Physiological mineralization is essential for proper development and function of hard tissues, such as bone, teeth, and growth plate cartilage, but it does not occur in soft tissues. Pathological ectopic mineralization, in contrast, occurs in soft tissues, including blood vessels, kidney, articular cartilage, and cardiovascular tissue. Here, we describe the simple method for detecting and measuring the presence of mineralized nodules in cardiac ventricular fibroblasts by using von Kossa and alizarin red S staining, and a colorimetric method for calcium quantification, respectively.

Calcium is one of the most important intracellular messengers in biological systems. Ca²⁺ microfluorimetry is a valuable tool to assess information about mechanisms involved in the regulation of intracellular Ca²⁺ levels in research on cells and in living tissues. In essence, the use of a dye that fluoresces in the presence of a target substance allows the detection of changes in the concentration of this molecule by determining the changes in the fluorescence of the probe (increases or decreases, depending on the nature of the dye used; for a review see Tsien et al. 1985). In this regard, there have been developed two different methodologies to assess intracellular Ca²⁺ measurements. On the one hand, ratiometric methods are based on the use of a ratio between two fluorescence intensities linked to the physicochemical properties of the probe. This allows correction of artifacts due to bleaching, changes in focus, variations in laser intensity, etc. but makes measurements and data processing more complicated since they require more expensive equipment with the possibility to change the wavelength emission/detection in a rapid way. Some ratiometric Ca²⁺ indicators are Fura-2 and Indo-1. On the other hand, on binding to Ca²⁺, indicators used for non-ratiometric measurements show a shift in their fluorescence intensity (the free indicator has usually a very weak fluorescence). Therefore, although an increase in fluorescence signal can be related directly to an increase in Ca²⁺ concentration, the fluorescence intensity depends on many factors such as acquisition conditions, probe concentration, optical path length, balance between the affinity constants of proteins binding Ca²⁺, among others. However, the fluxes of Ca²⁺ are of such a magnitude that these interferences are minor contributors to biases in the measurements. There are many non-ratiometric calcium indicators, some of which are Fluo-3, Fluo-4 and Calcium-Green-3. Consequently, the most suitable Ca²⁺-probe for each experiment will depend on the range of Ca²⁺ concentration that has to be evaluated, instrumentation, loading requirements, etc. In the present report we describe the protocol employed to quantify intracellular Ca²⁺ changes in peritoneal macrophages using Fura-2 as a fluorimetric probe and a microfluorimetric protocol that allows quantification of responding cells to a given stimulus, localization of the main intracellular domains sensing Ca²⁺ changes and a time-resolved analysis of the Fura-2 fluorescence that reflects the intracellular dynamics of Ca²⁺ in these cells (Través et al., 2013).