Histochemistry and Cell Biology

, Volume 123, Issue 1, pp 51–60 | Cite as

Fluorescently labeled inhibitors detect localized serine protease activities in Drosophila melanogaster pole cells, embryos, and ovarian egg chambers

  • Rasmus Kragh Jakobsen
  • Shin Ono
  • James C. Powers
  • Robert DeLotto
Original Paper


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.


Oogenesis 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.


  1. Abuelyaman AS, Hudig D, Woodard SL, Powers JC (1994) Fluorescent derivatives of diphenyl [1-(N-peptidylamino)alkyl]phosphonate esters: synthesis and use in the inhibition and cellular localization of serine proteases. Bioconjug Chem 5:400–405PubMedGoogle Scholar
  2. Adams MD, Celniker SE, Holt RA, Evans CA, Gocayne JD, Amanatides PG, Scherer SE, Li PW, Hoskins RA, Galle RF, et al (2000) The genome sequence of Drosophila melanogaster. Science 287:2185–2195CrossRefPubMedGoogle Scholar
  3. Bronner G, Jackle H (1991) Control and function of terminal gap gene activity in the posterior pole region of the Drosophila embryo. Mech Dev 35:205–211PubMedGoogle Scholar
  4. Campos-Ortega JA, Hartenstein V (1985) The embryonic development of Drosophila melanogaster. Springer, Berlin Heidelberg New YorkGoogle Scholar
  5. Casali A, Casanova J (2001) The spatial control of Torso RTK activation: a C-terminal fragment of the Trunk protein acts as a signal for Torso receptor in the Drosophila embryo. Development 128:1709–1715PubMedGoogle Scholar
  6. Chasan R, Jin Y, Anderson KV (1992) Activation of the Easter zymogen is regulated by five other genes to define dorsal-ventral polarity in the Drosophila embryo. Development 115:607–616PubMedGoogle Scholar
  7. Dissing M, Giordano H, DeLotto R (2001) Autoproteolysis and feedback in a protease cascade directing Drosophila dorsal-ventral cell fate. EMBO J 20:2387–2393PubMedGoogle Scholar
  8. Ephrussi A, Lehmann R (1992) Induction of germ cell formation by oskar. Nature 358:387–392PubMedGoogle Scholar
  9. Erlanger BF, Kokowsky N, Cohen W (1961) The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 95:271–278PubMedGoogle Scholar
  10. Fessler JH, Fessler LI (1989) Drosophila extracellular matrix. Annu Rev Cell Biol 5:309–339PubMedGoogle Scholar
  11. Furriols M, Casanova J (2003) In and out of Torso RTK signalling. EMBO J 22:1947–1952PubMedGoogle Scholar
  12. Gettins P, Patston PA, Schapira M (1992) Structure and mechanism of action of serpins. Hematol Oncol Clin North Am 6:1393–1408PubMedGoogle Scholar
  13. Hecht PM, Anderson KV (1992) Extracellular proteases and embryonic pattern formation. Trends Cell Biol 2:197–202PubMedGoogle Scholar
  14. King RC (1970) Ovarian development in Drosophila melanogaster. Academic, New YorkGoogle Scholar
  15. Kisiel W, Fujikawa K (1983) Enzymological aspects of blood coagulation. Behring Inst Mitt 73:29–42PubMedGoogle Scholar
  16. 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
  17. Lojda Z (1996) The use of substrates with 7-amino-3-trifluoromethylcoumarine (AFC) leaving group in the localization of protease activities in situ. Acta Histochem 98:215–228PubMedGoogle Scholar
  18. Milner JM, Elliott SF, Cawston TE (2001) Activation of procollagenases is a key control point in cartilage collagen degradation: interaction of serine and metalloproteinase pathways. Arthritis Rheum 44:2084–2096PubMedGoogle Scholar
  19. Neurath H (1986) The versatility of proteolytic enzymes. J Cell Biochem 32:35–49PubMedGoogle Scholar
  20. Neurath H (1991) Proteolytic processing and regulation. Enzyme 45:239–243PubMedGoogle Scholar
  21. Pino-Heiss S, Schubiger G (1989) Extracellular protease production by Drosophila imaginal discs. Dev Biol 132:282–291PubMedGoogle Scholar
  22. Powers JC, Asgian JL, Ekici OD, James KE (2002) Irreversible inhibitors of serine, cysteine, and threonine proteases. Chem Rev 102:4639–4750CrossRefPubMedGoogle Scholar
  23. Rubin GM (2000) Biological annotation of the Drosophila genome sequence. Novartis Found Symp 229:79–82PubMedGoogle Scholar
  24. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  25. Spradling AC (1993) Germline cysts: communes that work. Cell 72:649–651PubMedGoogle Scholar
  26. Stein D, Nusslein-Volhard C (1992) Multiple extracellular activities in Drosophila egg perivitelline fluid are required for establishment of embryonic dorsal-ventral polarity. Cell 68:429–440PubMedGoogle Scholar
  27. Stein D, Roth S, Vogelsang E, Nusslein-Volhard C (1991) The polarity of the dorsoventral axis in the Drosophila embryo is defined by an extracellular signal. Cell 65:725–735PubMedGoogle Scholar
  28. Swanson MM, Poodry CA (1980) Pole cell formation in Drosophila melanogaster. Dev Biol 75:419–430PubMedGoogle Scholar
  29. Tautz D, Pfeifle C (1989) A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98:81–85CrossRefPubMedGoogle Scholar
  30. Travis J, Guzdek A, Potempa J, Watorek W (1990) Serpins: structure and mechanism of action. Biol Chem Hoppe Seyler 371(suppl):3–11PubMedGoogle Scholar
  31. van der Meer JM, Jaffe LF (1983) Elemental composition of the perivitelline fluid in early Drosophila embryos. Dev Biol 95:249–252PubMedGoogle Scholar
  32. Verheyen E, Cooley L(1994) Looking at oogenesis. Methods Cell Biol 44:545–561PubMedGoogle Scholar
  33. Williams MJ (2001) Regulation of antibacterial and antifungal innate immunity in fruitflies and humans. Adv Immunol 79:225–259PubMedGoogle Scholar
  34. Zalokar M, Erk I (1976) Division and migration of nuclei during early embryogenesis of Drosophila melanogaster. J Micro Biol Cell 25:97–106Google Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Rasmus Kragh Jakobsen
    • 1
  • Shin Ono
    • 2
  • James C. Powers
    • 3
  • Robert DeLotto
    • 1
  1. 1.Department of Genetics, Institute of Molecular BiologyUniversity of CopenhagenCopenhagen KDenmark
  2. 2.Department of System Engineering of Materials and Life Science, Faculty of EngineeringToyama UniversityToyamaJapan
  3. 3.Department of Chemistry and BiochemistryGeorgia Institute of TechnologyAtlantaUSA

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