Chapter 4: Culturing and Visualizing Cells

Intracellular Ion Concentrations Can Be Determined with Ion-Sensitive Fluorescent Dyes

EXPERIMENTAL FIGURE 4-12 Fura-2, a Ca²⁺-sensitive fluorochrome, can be used to monitor the relative concentrations of cytosolic Ca²⁺ in different regions of live cells. (Left) In a moving leukocyte, a Ca²⁺ gradient is established. The highest concentrations (green) are at the rear of the cell, where cortical contractions take place, and the lowest concentrations (blue) are at the cell front, where actin undergoes polymerization. (Right) When a pipette filled with chemotactic molecules placed to the side of the cell induces the cell to turn, the Ca²⁺ concentration momentarily increases throughout the cytoplasm, and a new gradient is established. The gradient is oriented such that the region of lowest Ca²⁺ (blue) lies in the direction that the cell will turn, whereas a region of high Ca²⁺ (yellow) always forms at the site that will become the rear of the cell.

[From R. A. Brundage et al., 1991, Science 254:703; courtesy of F. Fay.]

The concentration of Ca²⁺ or H⁺ within live cells can be measured with the aid of fluorescent dyes, or fluorochromes, whose fluorescence depends on the concentration of these ions. As discussed in later chapters, intracellular Ca²⁺ and H⁺ concentrations have pronounced effects on many cellular processes. For instance, many hormones and other stimuli cause a rise in cytosolic Ca²⁺ from the resting level of about 10⁻⁷ M to 10⁻⁶ M, which induces various cellular responses such as the contraction of muscle.

The fluorochrome fura-2, which is sensitive to Ca²⁺, contains five carboxylate groups that form ester linkages with ethanol. The resulting fura-2 ester is lipophilic and can diffuse from the medium across the plasma membrane into cells. Within the cytosol, esterases hydrolyze the fura-2 ester, yielding fura-2, whose free carboxylate groups render the molecule nonlipophilic and thus unable to cross cellular membranes, so it remains in the cytosol. Inside cells, each fura-2 molecule can bind a single Ca²⁺ ion, but no other cellular cation. This binding, which is proportional to the cytosolic Ca²⁺ concentration over a certain range, increases the fluorescence of fura-2 at one particular wavelength. At a second wavelength, the fluorescence of fura-2 is the same whether or not Ca²⁺ is bound and thus provides a measure of the total amount of fura-2 in a region of the cell. By examining cells continuously in the fluorescence microscope and measuring rapid changes in the ratio of fura-2 fluorescence at the two wavelengths, one can quantify rapid changes in the fraction of fura-2 that has bound a Ca²⁺ ion, and thus in the concentration of cytosolic Ca²⁺ (Figure 4-12).

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Fluorescent dyes (e.g., SNARF-1) that are sensitive to H⁺ concentrations can be used similarly to monitor the cytosolic pH of live cells. Other useful probes consist of a fluorochrome linked to a weak base that is only partially protonated at neutral pH and thus can freely permeate cellular membranes. In acidic organelles, however, these probes become protonated; because the protonated probes cannot recross the organelle membrane, they accumulate in the lumen at concentrations much greater than in the cytosol. Thus this type of fluorescent dye can be used to specifically stain particular organelles in live cells (Figure 4-13).

EXPERIMENTAL FIGURE 4-13 Location of lysosomes and mitochondria in a cultured living bovine pulmonary artery endothelial cell. The cell was stained with a green-fluorescing dye that is specifically bound to mitochondria and a red-fluorescing dye that is specifically incorporated into lysosomes. The image was sharpened using a deconvolution computer program discussed later in the chapter. N, nucleus.