The role of gap junctional intercellular communication (GJIC) in regulation of normal growth and differentiation is becoming increasingly recognized as a major cellular function. GJIC consists of intercellular exchange of low molecular weight molecules, and is the only means for direct contact between cytoplasms of adjacent animal cells. Disturbances of GJIC have been associated with many pathological conditions, such as carcinogenesis or hereditary illness. Reliable and accurate methods for the determination of GJIC are therefore important in cell biology studies. There are several methods used successfully in numerous laboratories to measure GJIC both in vitro and in vivo. This review comments on techniques currently used to study cell-to-cell communication, either by measuring dye transfer, as in methods like microinjection, scrape loading, gap-fluorescence recovery after photobleaching (gap-FRAP), the preloading assay, and local activation of a molecular fluorescent probe (LAMP), or by measuring electrical conductance and metabolic cooperation. As we will discuss in this review, these techniques are not equivalent but instead provide complementary information. We will focus on their main advantages and limitations. Although biological applications guide the choice of techniques we describe, we also review points that must be taken into consideration before using a methodology, such as the number of cells to analyze.

Introduction

Most cells in multicellular organisms perform gap junctional intercellular communication (GJIC). This is the well-known form for direct contact between cytoplasms of adjacent animal cells. The juxtaposition of two half-channels, called connexons, constitutes this junction (Figure 1). Each connexon is inserted into the cytoplasmic membrane of two neighboring cells. It is made of six protein subunits called connexins (Cx) and can be homomeric or heteromeric (1). Connexons are absent from spermatocytes, erythrocytes, thrombocytes, adult skeletal muscle, and some neuronal subpopulations (2). The Cx subunit is a four-transmembrane spanning protein, harboring two extracellular loops, a cytoplasmic loop, and a cytoplasmatic N-as well as C-terminal region (Figure 1). The Cx family in humans is composed of 21 types of proteins classified according to their molecular weight. One cell type can express more than one Cx isoform (3). The flux of molecules through these channels includes the passive diffusion of small (<1 kDa) and hydrophilic molecules, such as metabolites (e.g., ATP), nutrients (e.g., glucose), and second messengers (e.g., triphosphate inositol, ionic coupling, and Ca²⁺) (4). GJIC was for a long time regarded as a relatively passive way of intercellular signaling through cell-to-cell tunnels; however, it was finally recognized that the degree of intercellular coupling is finely regulated in three main aspects: (i) the number of channels present in the membrane, (ii) their functional state, and (iii) their permeability (5). These channels are critical to several physiological roles, such as impulse propagation in the heart and neurons (6), response of tissues to hormones (7), regulation of embryonic development (8), homeostasis (9), and regulation of cellular proliferation (10).

Figure 1. Basic intercellular communication topology and gap junction structure. (Click to enlarge)

Gap junction channels can be regulated by several agents, including voltage, phosphorylation, intracellular calcium and pH, adhesion proteins, extracellular matrix, and hormones (11).

Studies of cell-to-cell communication are carried out either by measuring dye transfer using techniques such as microinjection, scrape loading, electroporation, gap-FRAP (fluorescence recovery after photobleaching), preloading assays, local activation of molecular fluorescent probe (LAMP), or by measuring electrical conductance and metabolic cooperation.

In this paper, we describe fluorescent dye transfer techniques and other methods commonly used to study GJIC capacity in vitro and in vivo. Currently, all of these techniques are widely used to understand the physiology of gap junctions; for instance, to study the modifications to their normal function by chemicals, carcinogenesis, cell growth, and embryo-genes, or to study the permeability and ion selectivity of Cx isoforms. Knowledge of reliable and accurate methods for the determination of GJIC functionality and permeability is therefore important in studies of cell biology.

Dye Transfer

Most of the methods available for investigating intercellular communication mediated by Cx channels are dependent on the introduction into living cells of nontoxic dyes that are traced in their eventual intercellular movements. Molecules suitable for such experiments should be small enough to cross gap junction channels, whose cutoff size is around 900 Da, and should not leak across a normal nonjunctional membrane. Most Cx channels are permeable to several tracers and until now, no dye has been shown to permeate only one type of gap junction channel, that is, to be specific for any Cx species. However, it is also clear that different Cx form channels with distinct permeabilities and that these specific properties may allow for subtle discrimination between molecules that differ only in size and/or electrical charge (12).

Microinjection

Principle

Microinjection of membrane-impermeable, nontoxic tracers has been the first technique used to identify and map the communication network of a wide variety of cell systems. Indeed, the existence of functional cell-to-cell channels can be verified by intercellular transfer of tracers, which requires intracellular injection of membrane-impermeable but gap junction– permeable dye (13).

The first cell-to-cell spread of a fluorescent diffusion tracer introduced into one cell by microinjection was demonstrated by Kanno and Loewenstein in 1964. A fluorescent dye within the micropipette (with a tip diameter about 0.2 µm) is injected intracellularly by an electrical or pressure pulse (Figure 2A). This technique introduces macromolecules into cells by a transient perturbation of the cell membrane, which does not affect cell viability (Table 1). If the adjacent cells are coupled by gap junctions, the dye will diffuse and label them. Fluorescein was the first fluorescent dye extensively used to characterize direct intercellular coupling (14). However, nonjunctional membrane was found to be permeable to this dye (15) and thus fluorescein was gradually replaced by Lucifer yellow (LY) (16). The choice of the most adequate tracer is dependent on several factors, including the scope of the experiment and the conditions under which the study is conducted.