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Detecting Caspase Activity in Drosophila Larval Imaginal Discs

  • Caitlin E. Fogarty
  • Andreas Bergmann
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1133)

Abstract

Caspases are a highly specialized class of cell death proteases. Since they are synthesized as inactive full-length zymogens, activation—at least of effector caspases and to some extent also of initiator caspases—requires a proteolytic cleavage event, generating a large and a small subunit, two of each forming the active caspase. The proteolytic cleavage event generates neo-epitopes at both the C-terminus of the large subunit and the N-terminus of the small subunit. The cleaved Caspase-3 (CC3) antibody was raised against the neo-epitope of the large subunit and thus detects only cleaved, but not full-length, Caspase-3. Although raised against human cleaved Caspase-3, the CC3 antibody cross-reacts in other species and detects cleaved caspases, most notably DrICE and Dcp-1, in Drosophila. This protocol describes the procedure for use of the CC3 antibody to detect caspase activity in larval imaginal discs in Drosophila.

Key words

Drosophila Cleaved-Caspase-3 Dronc DrICE Dcp-1 Cell death Non-apoptotic function Imaginal Disc Immunolabeling 

Notes

Acknowledgement

We would like to thank Yun Fan, Ernesto Perez, and Jillian Lindblad for their technical expertise and review of the manuscript. This work was supported by grants from the National Institutes of Health (GM068016 and GM107789).

References

  1. 1.
    Kumar S (2007) Caspase function in programmed cell death. Cell Death Differ 14(1):32–43. doi: 10.1038/sj.cdd.4402060 PubMedCrossRefGoogle Scholar
  2. 2.
    Xu D, Woodfield SE, Lee TV, Fan Y, Antonio C, Bergmann A (2009) Genetic control of programmed cell death (apoptosis) in Drosophila. Fly (Austin) 3(1):78–90CrossRefGoogle Scholar
  3. 3.
    Crawford ED, Seaman JE, Barber AE II, David DC, Babbitt PC, Burlingame AL, Wells JA (2012) Conservation of caspase substrates across metazoans suggests hierarchical importance of signaling pathways over specific targets and cleavage site motifs in apoptosis. Cell Death Differ 19(12):2040–2048. doi: 10.1038/cdd.2012.99 PubMedCrossRefGoogle Scholar
  4. 4.
    Xu D, Li Y, Arcaro M, Lackey M, Bergmann A (2005) The CARD-carrying caspase Dronc is essential for most, but not all, developmental cell death in Drosophila. Development 132(9): 2125–2134PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Geisbrecht ER, Montell DJ (2004) A role for Drosophila IAP1-mediated caspase inhibition in Rac-dependent cell migration. Cell 118(1): 111–125. doi: 10.1016/j.cell.2004.06.020 PubMedCrossRefGoogle Scholar
  6. 6.
    Suzanne M, Petzoldt AG, Speder P, Coutelis JB, Steller H, Noselli S (2010) Coupling of apoptosis and L/R patterning controls stepwise organ looping. Curr Biol 20(19): 1773–1778. doi: 10.1016/j.cub.2010.08.056 PubMedCrossRefGoogle Scholar
  7. 7.
    Kuranaga E, Matsunuma T, Kanuka H, Takemoto K, Koto A, Kimura K, Miura M (2011) Apoptosis controls the speed of looping morphogenesis in Drosophila male terminalia. Development 138(8):1493–1499. doi: 10.1242/dev.058958 PubMedCrossRefGoogle Scholar
  8. 8.
    Fan Y, Bergmann A (2008) Distinct mechanisms of apoptosis-induced compensatory proliferation in proliferating and differentiating tissues in the Drosophila eye. Dev Cell 14(3):399–410. doi: 10.1016/j.devcel.2008.01.003 PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Huh JR, Guo M, Hay BA (2004) Compensatory proliferation induced by cell death in the Drosophila wing disc requires activity of the apical cell death caspase Dronc in a nonapoptotic role. Curr Biol 14(14):1262–1266. doi: 10.1016/j.cub.2004.06.015 PubMedCrossRefGoogle Scholar
  10. 10.
    Kondo S, Senoo-Matsuda N, Hiromi Y, Miura M (2006) DRONC coordinates cell death and compensatory proliferation. Mol Cell Biol 26(19):7258–7268. doi: 10.1128/MCB.00183-06 PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Wells BS, Yoshida E, Johnston LA (2006) Compensatory proliferation in Drosophila imaginal discs requires Dronc-dependent p53 activity. Curr Biol 16(16):1606–1615. doi: 10.1016/j.cub.2006.07.046 PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Arama E, Agapite J, Steller H (2003) Caspase activity and a specific cytochrome C are required for sperm differentiation in Drosophila. Dev Cell 4(5):687–697PubMedCrossRefGoogle Scholar
  13. 13.
    Arama E, Bader M, Srivastava M, Bergmann A, Steller H (2006) The two Drosophila cytochrome C proteins can function in both respiration and caspase activation. EMBO J 25(1):232–243. doi: 10.1038/sj.emboj.7600920 PubMedCrossRefGoogle Scholar
  14. 14.
    Stoven S, Silverman N, Junell A, Hedengren-Olcott M, Erturk D, Engstrom Y, Maniatis T, Hultmark D (2003) Caspase-mediated processing of the Drosophila NF-kappaB factor Relish. Proc Natl Acad Sci U S A 100(10):5991–5996. doi: 10.1073/pnas.1035902100 PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Leulier F, Rodriguez A, Khush RS, Abrams JM, Lemaitre B (2000) The Drosophila caspase Dredd is required to resist gram-negative bacterial infection. EMBO Rep 1(4):353–358. doi: 10.1093/embo-reports/kvd073 PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Yacobi-Sharon K, Namdar Y, Arama E (2013) Alternative germ cell death pathway in Drosophila involves HtrA2/Omi, lysosomes, and a caspase-9 counterpart. Dev Cell 25(1): 29–42. doi: 10.1016/j.devcel.2013.02.002 PubMedCrossRefGoogle Scholar
  17. 17.
    Ciancio G, Pollack A, Taupier MA, Block NL, Irvin GL III (1988) Measurement of cell-cycle phase-specific cell death using Hoechst 33342 and propidium iodide: preservation by ethanol fixation. J Histochem Cytochem 36(9): 1147–1152PubMedCrossRefGoogle Scholar
  18. 18.
    Abrams JM, White K, Fessler LI, Steller H (1993) Programmed cell death during Drosophila embryogenesis. Development 117(1):29–43PubMedGoogle Scholar
  19. 19.
    Gavrieli Y, Sherman Y, Ben-Sasson SA (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119(3):493–501PubMedCrossRefGoogle Scholar
  20. 20.
    Chen P, Abrams JM (2000) Analysis of programmed cell death and apoptosis in Drosophila. Methods Enzymol 322:65–76PubMedCrossRefGoogle Scholar
  21. 21.
    Srinivasan A, Roth KA, Sayers RO, Shindler KS, Wong AM, Fritz LC, Tomaselli KJ (1998) In situ immunodetection of activated caspase-3 in apoptotic neurons in the developing nervous system. Cell Death Differ 5(12):1004–1016. doi: 10.1038/sj.cdd.4400449 PubMedCrossRefGoogle Scholar
  22. 22.
    Yu SY, Yoo SJ, Yang L, Zapata C, Srinivasan A, Hay BA, Baker NE (2002) A pathway of signals regulating effector and initiator caspases in the developing Drosophila eye. Development 129(13):3269–3278PubMedGoogle Scholar
  23. 23.
    Fan Y, Bergmann A (2010) The cleaved-Caspase-3 antibody is a marker of Caspase-9-like DRONC activity in Drosophila. Cell Death Differ 17(3):534–539. doi: 10.1038/cdd.2009.185 PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Florentin A, Arama E (2012) Caspase levels and execution efficiencies determine the apoptotic potential of the cell. J Cell Biol 196(4):513–527. doi: 10.1083/jcb.201107133 PubMedCrossRefGoogle Scholar
  25. 25.
    Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9(7):671–675PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Caitlin E. Fogarty
    • 1
  • Andreas Bergmann
    • 1
  1. 1.Department of Cancer BiologyUniversity of Massachusetts Medical SchoolWorcesterUSA

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