Résumé
The field of signal transduction has boomed in recent years. Since the discovery of tyrosine phosphorylation 15 years ago, more than 25 different families of tyrosine kinases have been described. Furthermore, many signal transduction pathways, initiated by ligand binding to receptors, and leading to growth, survival, or differentiation, depending on cell type and signal context, have been elucidated. Many investigators have sought to determine the functions of these signaling pathways, using a combination of biochemistry and molecular biology. For example, receptor tyrosine kinase mutants lacking binding sites for particular signaling molecules have been introduced into cells lacking wild-type receptors (see, for example, Rönnstrand et al. 1992; Valius and Kazlauskas 1993). However, the conclusions from this approach could be compromised if the receptor does not function identically in a heterologous cell type. In an approach that does not require the use of heterologous cell types, so-called dominant negative forms of signaling proteins (that presumably compete with endogenous protein for substrates and other effector molecules) are introduced into cells. Here the problem is that it may not be possible to derive stable cell lines that express such inhibitory proteins. Several laboratories, including our own, have circumvented these difficulties by using microinjection to introduce dominant negative mutants (and inhibitory antibodies) into single cells (Mulcahy et al. 1985; Riabowol et al. 1988; Twamley-Stein et al. 1993; Sasaoka et al. 1994; Roche et al. 1994, 1995a, b, 1996). We will describe this approach here.