Single-cell method for estimation of protein tyrosine kinases in the zebrafish egg

The zebrafish system has many features that are ideal for the study of signal transduction enzymes involved in fertilization. However, one problem that the zebrafish egg shares with eggs of many species is that most of the signal transduction proteins are already synthesized and stored in the egg before ovulation. This makes it difficult to modify the proteome of the egg by transfection or knockdown methods and leaves microinjection of exogenous proteins as the only alternative. In many cases, the researcher must test the effect of an exogenous molecule such as a dominant-negative fusion protein or a blocking antibody on fertilization and development. Although the zebrafish egg is amendable to microinjection, the number of eggs that can be injected is usually insufficient for the type of immunoprecipitation analysis used to detect most protein tyrosine kinases (PTKs). Methods for measurement of serine-threonine kinases have been developed for individual Xenopus laevis or mammalian eggs; however, these kinases are present in higher abundance than most PTKs. In an effort to find a solution to this problem, we have begun to develop methods to measure PTK activity in single zebrafish eggs. The general approach has been to prepare a particulate fraction from a single egg and then solubilize this crude fraction in a nonionic detergent and do an autophosphosphorylation assay using high-specific-activity [gamma-32P]ATP. When the reaction products are resolved on sodium dodecyl sulfate (SDS) gels and treated with strong alkali, autoradiography provides a crude measure of the PTK activity in the egg. This method does not use immunoprecipitation to select for a particular kinase because the large amount of yolk makes immunoprecipitation very unreliable. Without an immunoprecipitation step, we are only able to estimate total PTK activity. Despite this limitation, this methodology allows us to determine the effect of an injected protein or other reagent on the PTK activity in a single fertilized egg.