细胞生物学

Calcium signaling is an emerging mechanism by which bacteria respond to environmental cues. To measure the intracellular free-calcium concentration in bacterial cells, [Ca²⁺]_i, a simple spectrofluorometric method based on the chemical probe Fura 2-acetoxy methyl ester (Fura 2-AM) is here presented using Pseudomonad bacterial cells. This is an alternative and quantitative method that can be completed in a short period of time with low costs, and it does not require the induction of heterologously expressed protein-based probes like Aequorin. Furthermore, it is possible to verify the properties of membrane channels involved in Ca²⁺ entry from the extracellular matrix. This method is in particular valuable for measuring [Ca²⁺]_i in the range of 0.1-39.8 µM in small cells like those of prokaryotes.

Ca²⁺ is an essential signaling messenger in all eukariotic cells, playing a pivotal role in many cellular functions as cell growth control (differentiation, fertilization and apoptosis), secretion, gene expression, enzyme regulation, among many others. This basic premise includes trypanosomatids as Trypanosoma cruzi and various species of Leishmania, the causative agents of Chagas disease and leishmaniasis respectively, where intracellular Ca²⁺ concentration ([Ca²⁺]_i) has been demonstrated to be finely regulated. Nevertheless [Ca²⁺]_i has been difficult to measure because of its very low cytoplasmic concentration (typically around 50-100 nM), when compared to the large concentration in the outside milieu (around 2 mM in blood). The development of intracellular fluorescent Ca²⁺-sensitive indicators has been of paramount importance to achieve this goal. The success was based on the synthesis of acetoximethylated derivative precursors, which allow the fluorescent molecules typically composed of many hydrophilic carboxyl groups responsible for its high affinity Ca²⁺-binding (and therefore very hydrophilic), to easily cross the plasma membrane. Once in the cell interior, unspecific esterases split the hydrophobic moiety from the fluorescent backbone structure, releasing the carboxyl groups, transforming it in turn to the acid form of the molecule, which remain trapped in the cytoplasm and regain its ability to fluoresce in a Ca²⁺-dependent manner. Among them, Fura-2 is by far the most used, because it is a ratiometric (two different wavelength excitation and one emission) Ca²⁺ indicator with a Ca²⁺ affinity compatible with the [Ca²⁺]_i. This protocol essentially consists in loading exponential phase parasites with Fura-2 and recording changes in [Ca²⁺]_i by mean of a double wavelength spectrofluorometer. This technique allows the acquisition of valuable information about [Ca²⁺]_i changes in real time, as a consequence of diverse stimuli or changes in conditions, as addition of drugs or different natural modulators.

Calcium (Ca²⁺) imaging aims at investigating the dynamic changes in live cells of its concentration ([Ca²⁺]) in different pathophysiological conditions. Ca²⁺ is an ubiquitous and versatile intracellular signal that modulates a large variety of cellular functions thanks to a cell type-specific toolkit and a complex subcellular compartmentalization.

Many Ca²⁺ sensors are presently available (chemical and genetically encoded) that can be specifically targeted to different cellular compartments. Using these probes, it is now possible to monitor Ca²⁺ dynamics of living cells not only in the cytosol but also within specific organelles. The choice of a specific sensor depends on the experimental design and the spatial and temporal resolution required.

Here we describe the use of novel Förster resonance energy transfer (FRET)-based fluorescent Ca²⁺ probes to dynamically and quantitatively monitor the changes in cytosolic and mitochondrial [Ca²⁺] in a variety of cell types and experimental conditions. FRET-based sensors have the enormous advantage of being ratiometric, a feature that makes them particularly suitable for quantitative and in vivo applications.

The physiological importance of mitochondrial calcium uptake, observed in processes such as ATP production, intracellular calcium signaling, and apoptosis, makes desirable a simple, straightforward way of investigating this event with unambiguous results. The following protocol uses a calcium-sensitive, membrane-impermeable fluorophore to monitor extra-mitochondrial calcium levels in the presence of permeabilized mammalian cells harboring activated mitochondria.