Abstract
Apoptosis can be routinely characterized using biomolecular markers such as in the TUNEL and the annexin V assays or by using fluorescent caspase substrates. Apoptosis can also be semi-quantitatively characterized using microscopy, which targets morphological features such as cell rounding, nuclear condensation and fragmentation as well as cell membrane blebbing. This label-free approach provides a limited resolution for the evolution of these events in time and relies heavily on subjective identification of the morphological features. Here we propose a label-free assay based on surface plasmon resonance (SPR) detection of minute morphology changes occurring as a result of apoptosis induction in an endothelial cell model (EA.hy926). At first, annexin V assays confirmed that our cellular model was responsive to TRAIL over a 12-hour period. Then, we show that SPR allows accurate monitoring of apoptosis by measuring (1) the duration of the latency period during which the apoptotic signal is integrated by the initiator caspases and transmitted to the executioner caspases, (2) the rate of the execution phase in which death substrates are cleaved and morphological changes occur, and (3) the total extent of apoptosis. Using these parameters, we characterized the responses obtained with TRAIL (EA.hy926, HeLa, AD-293) and the anti-Fas antibody (HeLa) for the extrinsic pathways and UV exposure (HeLa) for the intrinsic pathways. By comparing the SPR time-course of apoptosis with phase contrast micrographs, we demonstrate that the cell morphological hallmarks of apoptosis are the major contributors to the SPR signal. Altogether, our results validate the use of SPR as an accurate label-free assay for the real-time monitoring of apoptosis-triggered cell morphological changes.
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Abbreviations
- SPR:
-
Surface plasmon resonance
- RVU:
-
Reflectance variation unit
- HBSS:
-
HEPES buffered salt solution
- TRAIL:
-
TNF (tumor necrosis factor)-related apoptosis-inducing ligand
- PE:
-
Phycoerythrin
- 7-AAD:
-
7-aminoactinomycin D
References
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
Feig C, Peter ME (2007) How apoptosis got the immune system in shape. Eur J Immunol 37(Suppl 1):S61–S70
O’Brien IE, Reutelingsperger CP, Holdaway KM (1997) Annexin-V and TUNEL use in monitoring the progression of apoptosis in plants. Cytometry 29(1):28–33
van Engeland M, Nieland LJ, Ramaekers FC, Schutte B, Reutelingsperger CP (1998) Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry 31(1):1–9
Heatwole VM (1999) TUNEL assay for apoptotic cells. Methods Mol Biol 115:141–148
Cen H, Mao F, Aronchik I, Fuentes RJ, Firestone GL (2008) DEVD-NucView488: a novel class of enzyme substrates for real-time detection of caspase-3 activity in live cells. FASEB J 22(7):2243–2252
Zhang HZ, Kasibhatla S, Guastella J, Tseng B, Drewe J, Cai SX (2003) N-Ac-DEVD-N′-(Polyfluorobenzoyl)-R110: novel cell-permeable fluorogenic caspase substrates for the detection of caspase activity and apoptosis. Bioconj Chem 14(2):458–463.
Coleman ML, Sahai ES, Yeo M, Bosch M, Dewar A, Olson MF (2001) Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I. Nat Cell Biol 3(4):339–345.
Ziegler U, Groscurth P (2004) Morphological features of cell death. News Physiol Sci 19:124–128
Fang Y, Li G, Ferrie AM (2007) Non-invasive optical biosensor for assaying endogenous G protein-coupled receptors in adherent cells. J Pharmacol Toxicol Methods 55(3):314–322
Scott CW, Peters MF (2010) Label-free whole-cell assays: expanding the scope of GPCR screening. Drug Discov Today 15(17–18):704–716
Boozer C, Kim G, Cong S, Guan H, Londergan T (2006) Looking towards label-free biomolecular interaction analysis in a high-throughput format: a review of new surface plasmon resonance technologies. Curr Opin Biotechnol 17(4):400–405
McDonnell JM (2001) Surface plasmon resonance: towards an understanding of the mechanisms of biological molecular recognition. Curr Opin Chem Biol 5(5):572–577
Pattnaik P (2005) Surface plasmon resonance: applications in understanding receptor–ligand interaction. Appl Biochem Biotechnol 126(2):79–92
Jason-Moller L, Murphy M, Bruno J (2006) Overview of biacore systems and their applications. Curr Protoc Protein Sci; Chapter 19:Unit 19.13
Danielson UH (2009) Fragment library screening and lead characterization using SPR biosensors. Curr Top Med Chem 9(18):1725–1735
de Mol NJ (2012) Surface plasmon resonance for proteomics. Methods Mol Biol 800:33–53.
Hide M, Tsutsui T, Sato H, Nishimura T, Morimoto K, Yamamoto S, Yoshizato K (2002) Real-time analysis of ligand-induced cell surface and intracellular reactions of living mast cells using a surface plasmon resonance-based biosensor. Anal Biochem 302(1):28–37.
Yanase Y, Suzuki H, Tsutsui T, Hiragun T, Kameyoshi Y, Hide M (2007) The SPR signal in living cells reflects changes other than the area of adhesion and the formation of cell constructions. Biosens Bioelectron 22(6):1081–1086
Suzuki H, Yanase Y, Tsutsui T, Ishii K, Hiragun T, Hide M (2008) Applying surface plasmon resonance to monitor the IgE-mediated activation of human basophils. Allergol Int 57(4):347–358
Robelek R, Wegener J (2010) Label-free and time-resolved measurements of cell volume changes by surface plasmon resonance (SPR) spectroscopy. Biosens Bioelectron 25(5):1221–1224
Tanaka M, Hiragun T, Tsutsui T, Yanase Y, Suzuki H, Hide M (2008) Surface plasmon resonance biosensor detects the downstream events of active PKCbeta in antigen-stimulated mast cells. Biosens Bioelectron 23(11):1652–1658
Lee SH, Ko HJ, Park TH (2009) Real-time monitoring of odorant-induced cellular reactions using surface plasmon resonance. Biosens Bioelectron 25(1):55–60
Cuerrier CM, Chabot V, Vigneux S, Aimez V, Escher E, Gobeil F, Charette PG, Grandbois M (2008) Surface plasmon resonance monitoring of cell monolayer integrity: implication of signaling pathways involved in actin-driven morphological remodeling. Cell Mol Bioeng 1(4):229–239.
Chabot V, Cuerrier CM, Escher E, Aimez V, Grandbois M, Charette PG (2009) Biosensing based on surface plasmon resonance and living cells. Biosens Bioelectron 24(6):1667–1673
Homola J (2003) Present and future of surface plasmon resonance biosensors. Anal Bioanal Chem 377(3):528–539.
Al-Rubeai M, Fussenegger M (eds) (2004) Cell engineering: apoptosis. Kluwer, Dordrecht
Sheridan JP, Marsters SA, Pitti RM, Gurney A, Skubatch M, Baldwin D, Ramakrishnan L, Gray CL, Baker K, Wood WI, Goddard AD, Godowski P, Ashkenazi A (1997) Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors. Science 277(5327):818–821
Wang S, El-Deiry WS (2003) TRAIL and apoptosis induction by TNF-family death receptors. Oncogene 22(53):8628–8633.
Kaufmann SH (ed) (1997) Apoptosis: pharmacological implications and therapeutic opportunities. Academic Press, San Diego
Melino G, Vaux D (eds) (2010) Cell Death. Wiley–Blackwell, Oxford
Boucher D, Blais V, Drag M, Denault JB (2011) Molecular determinants involved in activation of caspase 7. Biosci Rep 31(4):283–294
Rao L, Perez D, White E (1996) Lamin proteolysis facilitates nuclear events during apoptosis. J Cell Biol 135(6 Pt 1):1441–1455
Mashima T, Naito M, Noguchi K, Miller DK, Nicholson DW, Tsuruo T (1997) Actin cleavage by CPP-32/apopain during the development of apoptosis. Oncogene 14(9):1007–1012.
Hacker G (2000) The morphology of apoptosis. Cell Tissue Res 301(1):5–17
Wen LP, Fahrni JA, Troie S, Guan JL, Orth K, Rosen GD (1997) Cleavage of focal adhesion kinase by caspases during apoptosis. J Biol Chem 272(41):26056–26061
Mills JC, Lee VM, Pittman RN (1998) Activation of a PP2A-like phosphatase and dephosphorylation of tau protein characterize onset of the execution phase of apoptosis. J Cell Sci 111(Pt 5):625–636
Rosenblatt J, Raff MC, Cramer LP (2001) An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mechanism. Curr Biol 11(23):1847–1857
Acknowledgments
The authors would like to acknowledge Yannick Miron for his technical help with the SPR apparatus and Véronique Blais for her support with flow cytometry analyses. This research was supported by funds from the Canadian Institutes of Health Research (CIHR) and the Natural Sciences and Engineering Research Council of Canada (NSERC) to MG, LG and JBD.
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The authors declare no conflict of interests.
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Maltais, JS., Denault, JB., Gendron, L. et al. Label-free monitoring of apoptosis by surface plasmon resonance detection of morphological changes. Apoptosis 17, 916–925 (2012). https://doi.org/10.1007/s10495-012-0737-y
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DOI: https://doi.org/10.1007/s10495-012-0737-y