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Detection of Mitochondrial Caspase Activity in Real Time In Situ in Live Cells

Published online by Cambridge University Press:  01 August 2004

Yingpei Zhang ,
Catherine Haskins ,
Marisa Lopez-Cruzan ,
Jianhua Zhang ,
Victoria E. Centonze  and
Brian Herman
Yingpei Zhang
Affiliation:
Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
Catherine Haskins
Affiliation:
Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
Marisa Lopez-Cruzan
Affiliation:
Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
Jianhua Zhang
Affiliation:
Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
Victoria E. Centonze
Affiliation:
Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
Brian Herman
Affiliation:
Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
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Abstract

Apoptosis plays an important role in many physiological and pathological processes. The initiation and execution of the cell death program requires activation of multiple caspases in a stringently temporal order. Here we describe a method that allows real-time observation of caspase activation in situ in live cells based on fluorescent resonance energy transfer (FRET) measurement using the prism and reflector imaging spectroscopy system (PARISS). When a fusion protein consisting of CFP connected to YFP via an intervening caspase substrate that has been targeted to a specific subcellular location is excited with a light source whose wavelength matches the cyan fluorescent protein (CFP) excitation peak, the energy absorbed by the CFP fluorophore is not emitted as fluorescence. Instead, the excitation energy is absorbed by the nearby yellow fluorescent protein (YFP) fluorophore that is covalently linked to CFP through a short peptide containing the caspase substrate. Cleavage of the linker peptide by caspases results in loss of FRET due to the separation of CFP and YFP fluorophores. Using a mitochondrially targeted CFP–caspase 3 substrate–YFP construct (mC3Y), we demonstrate for the first time that there is caspase-3-like activity in the mitochondrial matrix of some cells at very late stage of apoptosis.

Keywords

PARISSFRETfluorescent spectral microscopycaspase activation
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 10 , Issue 4 , August 2004 , pp. 442 - 448
Copyright
© 2004 Microscopy Society of America

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References

REFERENCES

Budihardjo, I., Oliver, H., Lutter, M., Luo, X., & Wang, X. (1999). Biochemical pathways of caspase activation during apoptosis. Annu Rev Cell Dev Biol 15, 269–290.Google Scholar
Cande, C., Cohen, I., Daugas, E., Ravagnan, L., Larochette, N., Zamzami, N., & Kroemer, G. (2002). Apoptosis-inducing factor (AIF): A novel caspase-independent death effector released from mitochondria. Biochimie 84, 215–222.Google Scholar
Krajewski, S., Krajewska, M., Ellerby, L.M., Welsh, K., Xie, Z., Deveraux, Q.L., Salvesen, G.S., Bredesen, D.E., Rosenthal, R.E., Fiskum, G., & Reed, J.C. (1999). Release of caspase-9 from mitochondria during neuronal apoptosis and cerebral ischemia. Proc Natl Acad Sci USA 96, 5752–5757.Google Scholar
Li, H. & Yuan, J. (1999). Deciphering the pathways of life and death. Curr Opin Cell Biol 11, 261–266.Google Scholar
Shikama, Y., Miyashita, M.U.T., & Yamada, M. (2001). Comprehensive studies on subcellular localizations and cell death-inducing activities of eight GFP-tagged apoptosis-related caspases. Exp Cell Res 264, 315–325.Google Scholar
Susin, S. A., Lorenzo, H.K., Zamzami, N., Marzo, I., Brenner, C., Larochette, N., Prevost, M.C., Alzari, P.M., & Kroemer, G. (1999). Mitochondrial release of caspase-2 and -9 during the apoptotic process. J Exp Med 189, 381–394.Google Scholar
van Loo, G., Saelens, X., Matthijssens, F., Schotte, P., Beyaert, R., Declercq, W., & Vandenabeele, P. (2002). Caspases are not localized in mitochondria during life or death. Cell Death Differ 9, 1207–1211.Google Scholar
Zhang, Y. & Herman, B. (2002). Ageing and apoptosis. Mech Ageing Dev 123, 245–260.Google Scholar