Fluorescently labeled inhibitors detect localized serine protease activities in Drosophila melanogaster pole cells, embryos, and ovarian egg chambers
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Serine proteases are typically synthesized as proteolytically inactive zymogens that often become activated in a limited and highly localized manner. Consequently, determination of the spatial and temporal activation pattern of these molecules is of great importance to understanding the biological processes that they mediate. Until only recently, the tools to conveniently address the question of where and when serine proteases are active within complex tissues have been lacking. In order to detect spatially restricted serine protease activities in Drosophila embryos and ovaries we introduce a technique using fluorescent synthetic and protein-based inhibitors. With this approach we have detected a novel serine protease activity with a relative mobility of 37 kDa, localized to the surface of pole cells, the germ-line precursors, in embryos between nuclear cycles 11 and 14 in development. A second novel cell-specific protease activity was localized to the tissues of early gastrulating embryos. Microinjection of inhibitors into the perivitelline space of stage 2 embryos perturbed normal embryonic development. Fluorescein-conjugated chymotrypsin inhibitor and Bowman-Birk inhibitor labeled protease activity localized to the oocyte–somatic follicle cell interface of the developing egg chamber. Our results suggest that this technique holds promise to identify new spatially restricted activities in adult Drosophila tissues and developing embryos.
KeywordsOogenesis Embryonic patterning Chloromethyl ketone Phosphonate Zymogen activation
We would like to thank Yvonne DeLotto for excellent technical assistance with the microinjections and Jakob Winther for constructive experimental criticism. This work was supported by the Danish Natural Science Research Council, the Danish Cancer Fund, the Vera and Carl Johan Michaelsens Legacy, and the US National Science Foundation to R.D., and by grants from the National Institute of General Medical Sciences (grants GM54401 and GM61964) to J.C.P.
- Campos-Ortega JA, Hartenstein V (1985) The embryonic development of Drosophila melanogaster. Springer, Berlin Heidelberg New YorkGoogle Scholar
- King RC (1970) Ovarian development in Drosophila melanogaster. Academic, New YorkGoogle Scholar
- LeMosy EK, Tan YQ, Hashimoto C (2001) Activation of a protease cascade involved in patterning the Drosophila embryo. Proc Natl Acad Sci U S A 98:5055–5060Google Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
- Zalokar M, Erk I (1976) Division and migration of nuclei during early embryogenesis of Drosophila melanogaster. J Micro Biol Cell 25:97–106Google Scholar