Summary

In vivo Biosensor Tracks Ikke-apoptotisk Caspase aktivitet i Drosophila

Published: November 27, 2016
doi:

Summary

For at detektere sunde celler i hele dyr, der indeholder lave niveauer af caspaseaktivitet, blev den meget følsomme biosensor betegnet CaspaseTracker genereret for Drosophila. Caspase-afhængige biosensor aktivitet detekteres i langlivede raske celler i hele de indre organer af voksne dyr, der er opdrættet under optimerede betingelser i fravær af død stimuli.

Abstract

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.

Introduction

Caspaser er cysteinproteaser, som medierer apoptotisk celledød ved spaltning mange intracellulære proteiner efter centrale aspartatrester. For eksempel, initiator-caspaser aktivere effektor-caspaser, derepressere DNA nukleaser, spalter cytoskeletale komponenter, og ændre lipidsammensætning af cellemembraner til hurtigt demontere celler og stimulere deres anerkendelse og engulfment ved naboceller, som bortskaffer cellen lig. 1-4 Det anslås at milliarder af celler dø per dag i det menneskelige legeme, og apoptose er en vigtig mekanisme af kemoterapi-induceret tumor celledød. 5 et andet sæt af caspaser kan forårsage celledød ved distinkte ikke-apoptotiske processer til at stimulere medfødte immunitet. 6 Derfor, mest forskning i caspaser har fokuseret på deres pro-død funktioner.

Interessant første undersøgelsesresultater på området afslørede, at de samme caspaser ansvarlige for at fremme celledød har også ikke-død funktioner. Banebrydende undersøgelser har vist, at caspaser er involveret i forskellige cellulære funktioner i raske celler, herunder reguleringen af celledeling og migrering under embryogenese. 7-9 Caspaser er nødvendige for sædcelleantal individualisering i Drosophila 10,11, for at blokere en alternativ necroptotic celledød vej i mus 12,13, og for microRNA forarbejdning i C. elegans. 14,15 I måske de længste levede celler, neuroner, caspaser og andre apoptotisk maskiner er impliceret i reguleringen af neuronal aktivitet ved beskæring synaptiske slutninger, en proces menes være vigtigt at styrke andre synapser for indlæring og hukommelse. 16- 18. Det er muligt, at caspaser lette synaptisk beskæring af en type mini-apoptose af bittesmå neuronale projektioner uden hele celledød. 19 imidlertid caspaser kan have alternative funktioner ikke er relateret til apoptose-lignende begivenheder. 20,21 dobbeltrolles i liv og død er ikke enestående for caspaser; BCL-2 familie proteiner og cytochrom c har roller i cellulære energetik i sunde celler, men er også en del af kernen apoptosecyklus, der aktiveres af mange typer af celle stress. 22-25 Selvom det ikke er bevist, synes det logisk, at evolutionen har kædet dag -jobs til døden-jobs inden for de samme molekyler at sikre rettidig eliminering af uegnede eller uønskede celler.

På nuværende tidspunkt er de molekylære mekanismer i ikke-apoptotisk caspaseaktivitet ikke forstået, og omfanget af ikke-apoptotisk caspaseaktivitet under embryonisk udvikling og i voksent væv vides heller ikke. En stor udfordring er vanskeligheden ved at skelne dag-job fra død-job af caspaser. I modsætning til apoptose og pyroptosis, når caspase aktivitet forstærkes af et proteolytisk kaskade, der forventes at forekomme ved meget lavere niveauer af enzymatisk aktivitet, sandsynligvis under påvisning af mange tilgængelige techn dag-job caspaserologies.

Før arbejdet præsenteret her, andre har udviklet en række caspase biosensorer til forskellige formål. De SCAT biosensorer (f.eks ECFP-DEVD-Venus) hurtigt opdage realtid caspase aktivitet i dyrkede celler og animalske væv ved hjælp FRET. 26,27 Ved caspase spaltning, den nukleare målrettet GFP-delen af Apoliner (mCD8-RFP-DQVD- nucGFP) undergår subcellulær relocalization inden for minutter, når dens plasmamembran-tether spaltes af caspaser. 28 Tilsvarende ApoAlert-pCaspase3-sensor (NES-DEVD-YFP-NLS) relocalizes fra cytosolen til cellekernen ved caspase-spaltning. 29,30 Mere for nylig blev kromoforen i iCasper ødelagde manipuleret til at fluorescere ved spaltning ved caspaser, hvilket tillader påvisning af biosensor aktivitet i realtid i neuroner i Drosophila embryoner, men primært i forbindelse med udviklingstoksicitet celledød. 31 Caspase-afhængig død olfaktoriske neuroner under lagringen var demonstbedømt af immuno-påvisning af caspase-spaltet form af CPV biosensorer (f.eks mCD8-PARP-Venus). 32,33 vigtigt, blev den aktiverede form af caspase-3 påvises i fravær af celledød ved følsomme immunofarvningsprocedure i ryggen på dyrkede neuroner, og i soma hjælp af caspase-afhængig fluorescens af nukleare CellEvent reporter-farvestof, men vanskeligheder har man mødt på grund af foto-toksicitet, selvom celledød blev forsinket til efter rygsøjlen elimination. 19 er således behov for nye caspase biosensorer til påvisning og spore celler med basal caspase-aktivitet in vivo.

For at overvinde disse vanskeligheder, genereret vi en ny dual farve caspase biosensor, betegnet CaspaseTracker. Denne strategi kombinerer en modificeret version af Drosophila caspase-følsomme Apoliner biosensor 28 med Drosophila G-TRACE FRT rekombinasesystem 34 permanent mærke og spore celler in vivo. <sup> 35 Gal4-aktiverede G-TRACE system tillader meget lave niveauer af caspaser at aktivere CaspaseTracker, hvilket resulterer i RFP udtryk i cytoplasmaet og permanent nuklear målrettet GFP-ekspression i en celle, der nogensinde har oplevet caspase aktivitet. 35 Dette system kan mærke celler hele livet i hele dyr ved hjælp Drosophila melanogaster, en tractable og udbredte modelsystem til undersøgelse af caspaser og celledød. 36-38

Protocol

1. Fremstilling af CaspaseTracker Flies For at forberede CaspaseTracker (DQVD) flyver til forsøg, udføre denne indlæg: UBI-CaspaseTracker x G-TRACE (UAS-RFP, UAS-FLP, Ubi> Stop> GFP-NLS), ved at overføre 7-10 jomfru kvindelige (eller mand) fluer bærer caspase biosensor substrat mCD8-DIAP1-Gal4 drevet af ubiquitinpromotoren 35 sammen med det samme antal mænd (eller kvinde) G-TRACE fluer, som har den anden kromosom cyo balancer at undgå letalitet den homozygote kombinat…

Representative Results

Der er to vigtige komponenter, der tillader CaspaseTracker at detektere caspaseaktivitet i normale sunde celler (figur 1A). Den første af disse er en 146 aminosyre caspase-spaltelig polypeptid modelleret efter caspase biosensor Apoliner (figur 1b). 28 Dette polypeptid er afledt af DIAP1 (Drosophila inhibitor af apoptose) indeholdende en enkelt naturligt forekommende caspase site, der spaltes under apoptose typisk af caspase DrICE. <s…

Discussion

Her viser vi bygge- og indre funktioner i CaspaseTracker der letter påvisning af udbredt basal caspase aktivitet hos raske væv. De kritiske trin til påvisning ikke-apoptotisk caspase aktivitet in vivo er: 1) at generere fluer med biosensoren transgen, 2) at kontrollere caspase-specifikke reporter funktion med passende kontroller, 3) øve dissektion teknikker til at overholde alle interne organsystemer af voksne Drosophila, og 4) skelne biosensor aktivitet fra autofluorescerende artefakter.

<p cl…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Vi takker Polan Santos og Darren Obbard for Drosophila illustrationer i fig. 2a, Marcelo Jacobs-Lorena for brug af JHMRI insektbetingelser. Dette arbejde blev støttet af Life Science Research Foundation fællesskab (HLT), University Grants udvalg af Hong Kong AOE / B-07/99 (MCF), og NIH giver NS096677, NS037402 og NS083373 (JMH). Ho Lam Tang er en Shurl og Kay Curci Foundation Fellow af Life Sciences Research Foundation.

Materials

CONSUMABLES AND REAGENTS
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
EQUIPMENT
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

References

  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).
check_url/kr/53992?article_type=t

Play Video

Cite This Article
Tang, H. L., Tang, H. M., Fung, M. C., Hardwick, J. M. In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila. J. Vis. Exp. (117), e53992, doi:10.3791/53992 (2016).

View Video