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In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

Published: November 27th, 2016



1W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, 2School of Life Sciences, Chinese University of Hong Kong

To detect healthy cells in whole animals that contain low levels of caspase activity, the highly sensitive biosensor designated CaspaseTracker was generated for Drosophila. Caspase-dependent biosensor activity is detected in long-lived healthy cells throughout the internal organs of adult animals reared under optimized conditions in the absence of death stimuli.

Caspases are the key mediators of apoptotic cell death via their proteolytic activity. When caspases are activated in cells to levels detectable by available technologies, apoptosis is generally assumed to occur shortly thereafter. Caspases can cleave many functional and structural components to cause rapid and complete cell destruction within a few minutes. However, accumulating evidence indicates that in normal healthy cells the same caspases have other functions, presumably at lower enzymatic levels. Studies of non-apoptotic caspase activity have been hampered by difficulties with detecting low levels of caspase activity and with tracking ultimate cell fate in vivo. Here, we illustrate the use of an ultrasensitive caspase reporter, CaspaseTracker, which permanently labels cells that have experienced caspase activity in whole animals. This in vivo dual color CaspaseTracker biosensor for Drosophila melanogaster transiently expresses red fluorescent protein (RFP) to indicate recent or on-going caspase activity, and permanently expresses green fluorescent protein (GFP) in cells that have experienced caspase activity at any time in the past yet did not die. Importantly, this caspase-dependent in vivo biosensor readily reveals the presence of non-apoptotic caspase activity in the tissues of organ systems throughout the adult fly. This is demonstrated using whole mount dissections of individual flies to detect biosensor activity in healthy cells throughout the brain, gut, malpighian tubules, cardia, ovary ducts and other tissues. CaspaseTracker detects non-apoptotic caspase activity in long-lived cells, as biosensor activity is detected in adult neurons and in other tissues at least 10 days after caspase activation. This biosensor serves as an important tool to uncover the roles and molecular mechanisms of non-apoptotic caspase activity in live animals.

Caspases are cysteine proteases that mediate apoptotic cell death by cleaving many intracellular proteins after key aspartate residues. For example, initiator caspases activate effector caspases, derepress DNA nucleases, cleave cytoskeletal components and alter the lipid composition of cell membranes to rapidly dismantle cells and stimulate their recognition and engulfment by neighboring cells that dispose of the cell corpses.1-4 It is estimated that billions of cells die per day in the human body, and apoptosis is an important mechanism of chemotherapy-induced tumor cell death.5 A different set of caspases can cause cell death by distinct non-ap....

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1. Preparation of CaspaseTracker Flies

  1. To prepare CaspaseTracker (DQVD) flies for experiments, perform this cross: UBI-CaspaseTracker x G-TRACE (UAS-RFP; UAS-FLP; Ubi>Stop>GFP-nls), by transferring 7-10 virgin female (or male) flies carrying the caspase biosensor substrate mCD8-DIAP1-Gal4 driven by the ubiquitin promoter35 together with the same number of male (or female) G-TRACE flies, which have the second chromosome CyO balancer to avoid lethality of the homozygous comb.......

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There are two key components that allow CaspaseTracker to detect caspase activity in normal healthy cells (Figure 1a). The first of these is a 146 amino acid caspase-cleavable polypeptide modeled after the caspase biosensor Apoliner (Figure 1b).28 This polypeptide is derived from DIAP1 (Drosophila inhibitor of apoptosis) containing a single naturally occurring caspase site that is cleaved during apoptosis typically by the caspase DrICE.......

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Here we illustrate the construction and inner workings of CaspaseTracker that facilitate detection of widespread basal caspase activity in healthy tissues. The critical steps for detecting non-apoptotic caspase activity in vivo are: 1) generating flies with the biosensor transgene, 2) verifying caspase-specific reporter function with appropriate controls, 3) practicing dissection techniques to observe all internal organ systems of adult Drosophila, and 4) distinguishing biosensor activity from autofluor.......

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We thank Polan Santos and Darren Obbard for Drosophila illustrations in Fig. 2a, Marcelo Jacobs-Lorena for use of the JHMRI insectary. This work was supported by the Life Science Research Foundation fellowship (H.L.T.), University Grants Committee of the Hong Kong AoE/B-07/99 (M.C.F.), and NIH grants NS096677, NS037402 and NS083373 (J.M.H.). Ho Lam Tang is a Shurl and Kay Curci Foundation Fellow of the Life Sciences Research Foundation.


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Name Company Catalog Number Comments
Vectashield Vector Products H-1000 Mounting medium
Forceps Ted Pella #505 (110mm, #5) Dumont tweezer biology grade, stainless steel
Hanging Drop Slides Fisher Scientific 12-565B Glass slides
Hoechst 33342 Molecular Probes H1399 DNA stain
Alexa Fluor 633 Phalloidin Molecular Probes A22284 Actin stain
Rat-Elav-7E8A10 anti-elav antibody  Developmental Studies Hybridoma Bank (DSHB) Antibody Registry ID:  AB_528218  Stain for Drosophla pan-neuronal ELAV
Cleaved caspase-3 (Asp175) antibody Cell Signaling Technology #9661 Stain for active fragment of caspase-3
ProLong Gold antifade reagent Life Technologies P36934 to preserve fluorophores 
ProLong Diamond Antifade Mountant Life Technologies P36961 to preserve fluorophores 
SylGard 182 Silicone Elastomer Kit Dow Corning  Product code: 0001023934 for dissection plates
LSM780 confocal microscope Carl Zeiss N/A Imaging
Carl Zeiss Stereomicroscope Stemi 2000  Carl Zeiss N/A Drosophila dissection
AmScope Fiber Optic Dual Gooseneck Microscope Illuminator, 150W AmScope WBM99316  Light source

  1. Salvesen, G. S., Abrams, J. M. Caspase activation - stepping on the gas or releasing the brakes? Lessons from humans and flies. Oncogene. 23, 2774-2784 (2004).
  2. Hay, B. A., Guo, M. Caspase-dependent cell death in Drosophila. Annu Rev Cell Dev Biol. 22, 623-650 (2006).
  3. Segawa, K., et al. Caspase-mediated cleavage of phospholipid flippase for apoptotic phosphatidylserine exposure. Science. 344, 1164-1168 (2014).
  4. Akagawa, H., et al. The role of the effector caspases drICE and dcp-1 for cell death and corpse clearance in the developing optic lobe in Drosophila. Dev Biol. , (2015).
  5. Souers, A. J., et al. ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets. Nat Med. 19, 202-208 (2013).
  6. Lamkanfi, M., Dixit, V. M. Mechanisms and functions of inflammasomes. Cell. 157, 1013-1022 (2014).
  7. Bergmann, A., Steller, H. Apoptosis, stem cells, and tissue regeneration. Sci Signal. 3, re8 (2010).
  8. Hyman, B. T., Yuan, J. Apoptotic and non-apoptotic roles of caspases in neuronal physiology and pathophysiology. Nat Rev Neurosci. 13, 395-406 (2012).
  9. Juraver-Geslin, H. A., Durand, B. C. Early development of the neural plate: new roles for apoptosis and for one of its main effectors caspase-3. Genesis. 53, 203-224 (2015).
  10. Arama, E., Agapite, J., Steller, H. Caspase activity and a specific cytochrome C are required for sperm differentiation in Drosophila. Dev Cell. 4, 687-697 (2003).
  11. Kaplan, Y., Gibbs-Bar, L., Kalifa, Y., Feinstein-Rotkopf, Y., Arama, E. Gradients of a ubiquitin E3 ligase inhibitor and a caspase inhibitor determine differentiation or death in spermatids. Dev Cell. 19, 160-173 (2010).
  12. Kaiser, W. J., et al. RIP3 mediates the embryonic lethality of caspase-8-deficient mice. Nature. 471, 368-372 (2011).
  13. Gunther, C., et al. Caspase-8 regulates TNF-alpha-induced epithelial necroptosis and terminal ileitis. Nature. 477, 335-339 (2011).
  14. Weaver, B. P., et al. CED-3 caspase acts with miRNAs to regulate non-apoptotic gene expression dynamics for robust development in C. elegans. Elife. 3, e04265 (2014).
  15. Ge, X., et al. A novel mechanism underlies caspase-dependent conversion of the dicer ribonuclease into a deoxyribonuclease during apoptosis. Cell Res. 24, 218-232 (2014).
  16. Fannjiang, Y., et al. BAK alters neuronal excitability and can switch from anti- to pro-death function during postnatal development. Dev Cell. 4, 575-585 (2003).
  17. Ofengeim, D., et al. N-terminally cleaved Bcl-xL mediates ischemia-induced neuronal death. Nat Neurosci. 15, 574-580 (2012).
  18. Li, Z., Sheng, M. Caspases in synaptic plasticity. Mol Brain. 5, 15 (2012).
  19. Erturk, A., Wang, Y., Sheng, M. Local pruning of dendrites and spines by caspase-3-dependent and proteasome-limited mechanisms. J Neurosci. 34, 1672-1688 (2014).
  20. Campbell, D. S., Okamoto, H. Local caspase activation interacts with Slit-Robo signaling to restrict axonal arborization. J Cell Biol. 203, 657-672 (2013).
  21. Feinstein-Rotkopf, Y., Arama, E. Can't live without them, can live with them: roles of caspases during vital cellular processes. Apoptosis. 14, 980-995 (2009).
  22. Li, P., et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 91, 479-489 (1997).
  23. Lewis, J., et al. Inhibition of virus-induced neuronal apoptosis by Bax. Nat Med. 5, 832-835 (1999).
  24. Chen, Y. B., et al. Bcl-xL regulates mitochondrial energetics by stabilizing the inner membrane potential. J Cell Biol. 195, 263-276 (2011).
  25. Yi, C. H., et al. Metabolic regulation of protein N-alpha-acetylation by Bcl-xL promotes cell survival. Cell. 146, 607-620 (2011).
  26. Takemoto, K., Nagai, T., Miyawaki, A., Miura, M. Spatio-temporal activation of caspase revealed by indicator that is insensitive to environmental effects. J Cell Biol. 160, 235-243 (2003).
  27. Takemoto, K., et al. Local initiation of caspase activation in Drosophila salivary gland programmed cell death in vivo. Proc Natl Acad Sci U S A. 104, 13367-13372 (2007).
  28. Bardet, P. L., et al. A fluorescent reporter of caspase activity for live imaging. Proc Natl Acad Sci U S A. 105, 13901-13905 (2008).
  29. Tang, H. L., et al. Cell survival, DNA damage, and oncogenic transformation after a transient and reversible apoptotic response. Mol Biol Cell. 23, 2240-2252 (2012).
  30. Golbs, A., Nimmervoll, B., Sun, J. J., Sava, I. E., Luhmann, H. J. Control of programmed cell death by distinct electrical activity patterns. Cereb Cortex. 21, 1192-1202 (2011).
  31. To, T. L., et al. Rationally designed fluorogenic protease reporter visualizes spatiotemporal dynamics of apoptosis in vivo. Proc Natl Acad Sci U S A. 112, 3338-3343 (2015).
  32. Florentin, A., Arama, E. Caspase levels and execution efficiencies determine the apoptotic potential of the cell. J Cell Biol. 196, 513-527 (2012).
  33. Chihara, T., et al. Caspase inhibition in select olfactory neurons restores innate attraction behavior in aged Drosophila. PLoS Genet. 10, e1004437 (2014).
  34. Evans, C. J., et al. G-TRACE: rapid Gal4-based cell lineage analysis in Drosophila. Nat Methods. 6, 603-605 (2009).
  35. Tang, H. L., Tang, H. M., Fung, M. C., Hardwick, J. M. In vivo CaspaseTracker biosensor system for detecting anastasis and non-apoptotic caspase activity. Sci Rep. 5, 9015 (2015).
  36. Suzanne, M., Steller, H. Shaping organisms with apoptosis. Cell Death Differ. 20, 669-675 (2013).
  37. Jenkins, V. K., Timmons, A. K., McCall, K. Diversity of cell death pathways: insight from the fly ovary. Trends Cell Biol. 23, 567-574 (2013).
  38. Sarkissian, T., Timmons, A., Arya, R., Abdelwahid, E., White, K. Detecting apoptosis in Drosophila tissues and cells. Methods. 68, 89-96 (2014).
  39. Williamson, W. R., Hiesinger, P. R. Preparation of developing and adult Drosophila brains and retinae for live imaging. J Vis Exp. , (2010).
  40. Wong, L. C., Schedl, P. Dissection of Drosophila ovaries. J Vis Exp. , e52 (2006).
  41. Tauc, H. M., Tasdogan, A., Pandur, P. Isolating intestinal stem cells from adult Drosophila midguts by FACS to study stem cell behavior during aging. J Vis Exp. , e52223 (2014).
  42. Ditzel, M., et al. Degradation of DIAP1 by the N-end rule pathway is essential for regulating apoptosis. Nat Cell Biol. 5, 467-473 (2003).
  43. Li, X., Wang, J., Shi, Y. Structural mechanisms of DIAP1 auto-inhibition and DIAP1-mediated inhibition of drICE. Nat Commun. 2, 408 (2011).
  44. Yi, S. X., Moore, C. W., Lee, R. E. Rapid cold-hardening protects Drosophila melanogaster from cold-induced apoptosis. Apoptosis. 12, 1183-1193 (2007).
  45. Drummond-Barbosa, D., Spradling, A. C. Stem cells and their progeny respond to nutritional changes during Drosophila oogenesis. Dev Biol. 231, 265-278 (2001).
  46. Fan, Y., Bergmann, A. The cleaved-Caspase-3 antibody is a marker of Caspase-9-like DRONC activity in Drosophila. Cell Death Differ. 17, 534-539 (2010).
  47. Fogarty, C. E., Bergmann, A. Detecting caspase activity in Drosophila larval imaginal discs. Methods Mol Biol. 1133, 109-117 (2014).
  48. Koushika, S. P., Lisbin, M. J., White, K. ELAV, a Drosophila neuron-specific protein, mediates the generation of an alternatively spliced neural protein isoform. Curr Biol. 6, 1634-1641 (1996).
  49. Albeck, J. G., et al. Quantitative analysis of pathways controlling extrinsic apoptosis in single cells. Mol Cell. 30, 11-25 (2008).
  50. Galluzzi, L., et al. Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes. Cell Death Differ. 16, 1093-1107 (2009).
  51. Holland, A. J., Cleveland, D. W. Chromoanagenesis and cancer: mechanisms and consequences of localized, complex chromosomal rearrangements. Nat Med. 18, 1630-1638 (2012).
  52. Green, D. R. . Means to an end : apoptosis and other cell death mechanisms. , (2011).
  53. Chau, B. N., et al. Signal-dependent protection from apoptosis in mice expressing caspase-resistant Rb. Nat Cell Biol. 4, 757-765 (2002).
  54. Lin, Y., Devin, A., Rodriguez, Y., Liu, Z. G. Cleavage of the death domain kinase RIP by caspase-8 prompts TNF-induced apoptosis. Genes Dev. 13, 2514-2526 (1999).
  55. Han, M. H., et al. The novel caspase-3 substrate Gap43 is involved in AMPA receptor endocytosis and long-term depression. Mol Cell Proteomics. 12, 3719-3731 (2013).
  56. Nakagawa, A., Shi, Y., Kage-Nakadai, E., Mitani, S., Xue, D. Caspase-dependent conversion of Dicer ribonuclease into a death-promoting deoxyribonuclease. Science. 328, 327-334 (2010).

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