Abstract

Intracellular protein transport and localization to subcellular regions are processes necessary for normal protein function. Fluorescent proteins can be fused to proteins of interest to track movement and determine localization within a cell. Currently, fluorescence microscopy combined with image processing is most often used to study protein movement and subcellular localization. In this contribution we evaluate a high-throughput time-resolved flow cytometry approach to correlate intracellular localization of human LC3 protein with the fluorescence lifetime of enhanced green fluorescent protein (EGFP). Subcellular LC3 localization to autophagosomes is a marker of the cellular process called autophagy. In breast cancer cells expressing native EGFP and EGFP-LC3 fusion proteins, we measured the fluorescence intensity and lifetime of (i) diffuse EGFP (ii) punctate EGFP-LC3 and (iii) diffuse EGFP-ΔLC3 after amino acid starvation to induce autophagy-dependent LC3 localization. We verify EGFP-LC3 localization with low-throughput confocal microscopy and compare to fluorescence intensity measured by standard flow cytometry. Our results demonstrate that time-resolved flow cytometry can be correlated to subcellular localization of EGFP fusion proteins by measuring changes in fluorescence lifetime.

© 2013 OSA

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  36. T. Nakabayashi, I. Nagao, M. Kinjo, Y. Aoki, M. Tanaka, and N. Ohta, “Stress-induced environmental changes in a single cell as revealed by fluorescence lifetime imaging,” Photochem. Photobiol. Sci.7(6), 671–674 (2008).
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  37. K. Elgass, K. Caesar, F. Schleifenbaum, Y. D. Stierhof, A. J. Meixner, and K. Harter, “Novel application of fluorescence lifetime and fluorescence microscopy enables quantitative access to subcellular dynamics in plant cells,” PLoS ONE4(5), e5716 (2009).
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  38. R. Pepperkok, A. Squire, S. Geley, and P. I. Bastiaens, “Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy,” Curr. Biol.9(5), 269–274 (1999).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  45. J. Liang, J. Zubovitz, T. Petrocelli, R. Kotchetkov, M. K. Connor, K. Han, J. H. Lee, S. Ciarallo, C. Catzavelos, R. Beniston, E. Franssen, and J. M. Slingerland, “PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest,” Nat. Med.8(10), 1153–1160 (2002).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]

2013 (3)

M. Zhao, P. G. Schiro, J. S. Kuo, K. M. Koehler, D. E. Sabath, V. Popov, Q. Feng, and D. T. Chiu, “An automated high-throughput counting method for screening circulating tumor cells in peripheral blood,” Anal. Chem.85(4), 2465–2471 (2013).
[CrossRef] [PubMed]

H. J. Yoo, J. Park, and T. H. Yoon, “High throughput cell cycle analysis using microfluidic image cytometry (μFIC),” Cytometry A83(4), 356–362 (2013).
[CrossRef] [PubMed]

R. Cao, V. Pankayatselvan, and J. P. Houston, “Cytometric sorting based on the fluorescence lifetime of spectrally overlapping signals,” Opt. Express21(12), 14816–14831 (2013).
[CrossRef] [PubMed]

2012 (4)

N. S. Barteneva, E. Fasler-Kan, and I. A. Vorobjev, “Imaging flow cytometry: coping with heterogeneity in biological systems,” J. Histochem. Cytochem.60(10), 723–733 (2012).
[PubMed]

J. R. Swedlow, “Innovation in biological microscopy: current status and future directions,” Bioessays34(5), 333–340 (2012).
[CrossRef] [PubMed]

J. P. Houston, M. A. Naivar, and J. P. Freyer, “Capture of fluorescence decay times by flow cytometry,” Curr. Protoc. Cytom. (2012).
[CrossRef]

A. Pliss, L. Zhao, T. Y. Ohulchanskyy, J. Qu, and P. N. Prasad, “Fluorescence lifetime of fluorescent proteins as an intracellular environment probe sensing the cell cycle progression,” ACS Chem. Biol.7(8), 1385–1392 (2012).
[CrossRef] [PubMed]

2011 (4)

M. C. Hung and W. Link, “Protein localization in disease and therapy,” J. Cell Sci.124(20), 3381–3392 (2011).
[CrossRef] [PubMed]

E. R. Tkaczyk and A. H. Tkaczyk, “Multiphoton flow cytometry strategies and applications,” Cytometry A79(10), 775–788 (2011).
[CrossRef] [PubMed]

R. Mathew and E. White, “Autophagy in tumorigenesis and energy metabolism: friend by day, foe by night,” Curr. Opin. Genet. Dev.21(1), 113–119 (2011).
[CrossRef] [PubMed]

P. Hundeshagen, A. Hamacher-Brady, R. Eils, and N. R. Brady, “Concurrent detection of autolysosome formation and lysosomal degradation by flow cytometry in a high-content screen for inducers of autophagy,” BMC Biol.9(1), 38 (2011).
[CrossRef] [PubMed]

2010 (3)

J. P. Houston, M. A. Naivar, P. Jenkins, and J. P. Freyer, “Digital Analysis and Sorting of Fluorescence Lifetime by Flow Cytometry,” Cytometry A77(9), 861–872 (2010).
[CrossRef] [PubMed]

R. F. Murphy, “Communicating subcellular distributions,” Cytometry A77(7), 686–692 (2010).
[CrossRef] [PubMed]

N. Mizushima, T. Yoshimori, and B. Levine, “Methods in Mammalian Autophagy Research,” Cell140(3), 313–326 (2010).
[CrossRef] [PubMed]

2009 (5)

T. Ito, S. Oshita, T. Nakabayashi, F. Sun, M. Kinjo, and N. Ohta, “Fluorescence lifetime images of green fluorescent protein in HeLa cells during TNF-alpha induced apoptosis,” Photochem. Photobiol. Sci.8(6), 763–767 (2009).
[CrossRef] [PubMed]

K. Elgass, K. Caesar, F. Schleifenbaum, Y. D. Stierhof, A. J. Meixner, and K. Harter, “Novel application of fluorescence lifetime and fluorescence microscopy enables quantitative access to subcellular dynamics in plant cells,” PLoS ONE4(5), e5716 (2009).
[CrossRef] [PubMed]

G. S. Elliott, “Moving pictures: imaging flow cytometry for drug development,” Comb. Chem. High Throughput Screen.12(9), 849–859 (2009).
[CrossRef] [PubMed]

E. A. Corcelle, P. Puustinen, and M. Jäättelä, “Apoptosis and autophagy: Targeting autophagy signalling in cancer cells -‘trick or treats’?” FEBS J.276(21), 6084–6096 (2009).
[CrossRef] [PubMed]

E. Shvets and Z. Elazar, “Flow cytometric analysis of autophagy in living mammalian cells,” Methods Enzymol.452, 131–141 (2009).
[CrossRef] [PubMed]

2008 (7)

I. M. Chu, L. Hengst, and J. M. Slingerland, “The Cdk inhibitor p27 in human cancer: prognostic potential and relevance to anticancer therapy,” Nat. Rev. Cancer8(4), 253–267 (2008).
[CrossRef] [PubMed]

F. Cecconi and B. Levine, “The role of autophagy in mammalian development: cell makeover rather than cell death,” Dev. Cell15(3), 344–357 (2008).
[CrossRef] [PubMed]

B. Seefeldt, R. Kasper, T. Seidel, P. Tinnefeld, K. J. Dietz, M. Heilemann, and M. Sauer, “Fluorescent proteins for single-molecule fluorescence applications,” J Biophotonics1(1), 74–82 (2008).
[CrossRef] [PubMed]

T. Nakabayashi, H. P. Wang, M. Kinjo, and N. Ohta, “Application of fluorescence lifetime imaging of enhanced green fluorescent protein to intracellular pH measurements,” Photochem. Photobiol. Sci.7(6), 668–670 (2008).
[CrossRef] [PubMed]

T. Nakabayashi, I. Nagao, M. Kinjo, Y. Aoki, M. Tanaka, and N. Ohta, “Stress-induced environmental changes in a single cell as revealed by fluorescence lifetime imaging,” Photochem. Photobiol. Sci.7(6), 671–674 (2008).
[CrossRef] [PubMed]

I. Tanida, T. Yamaji, T. Ueno, S. Ishiura, E. Kominami, and K. Hanada, “Consideration about negative controls for LC3 and expression vectors for four colored fluorescent protein-LC3 negative controls,” Autophagy4(1), 131–134 (2008).
[PubMed]

A. R. Kristensen, S. Schandorff, M. Høyer-Hansen, M. O. Nielsen, M. Jäättelä, J. Dengjel, and J. S. Andersen, “Ordered organelle degradation during starvation-induced autophagy,” Mol. Cell. Proteomics7(12), 2419–2428 (2008).
[CrossRef] [PubMed]

2007 (3)

M. A. Naivar, J. D. Parson, M. E. Wilder, R. C. Habbersett, B. S. Edwards, L. Sklar, J. P. Nolan, S. W. Graves, J. C. Martin, J. H. Jett, and J. P. Freyer, “Open, reconfigurable cytometric acquisition system: ORCAS,” Cytometry A71(11), 915–924 (2007).
[CrossRef] [PubMed]

A. Kuma, M. Matsui, and N. Mizushima, “LC3, an autophagosome marker, can be incorporated into protein aggregates independent of autophagy: caution in the interpretation of LC3 localization,” Autophagy3(4), 323–328 (2007).
[PubMed]

S. Kimura, T. Noda, and T. Yoshimori, “Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3,” Autophagy3(5), 452–460 (2007).
[PubMed]

2005 (3)

A. Esposito, H. C. Gerritsen, and F. S. Wouters, “Fluorescence lifetime heterogeneity resolution in the frequency domain by lifetime moments analysis,” Biophys. J.89(6), 4286–4299 (2005).
[CrossRef] [PubMed]

E. T. W. Bampton, C. G. Goemans, D. Niranjan, N. Mizushima, and A. M. Tolkovsky, “The dynamics of autophagy visualized in live cells - From autophagosome formation to fusion with endo/lysosomes,” Autophagy1(1), 23–37 (2005).
[CrossRef] [PubMed]

B. Treanor, P. M. Lanigan, K. Suhling, T. Schreiber, I. Munro, M. A. Neil, D. Phillips, D. M. Davis, and P. M. French, “Imaging fluorescence lifetime heterogeneity applied to GFP-tagged MHC protein at an immunological synapse,” J. Microsc.217(1), 36–43 (2005).
[CrossRef] [PubMed]

2004 (3)

A. H. Clayton, Q. S. Hanley, and P. J. Verveer, “Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data,” J. Microsc.213(1), 1–5 (2004).
[CrossRef] [PubMed]

I. Tanida, T. Ueno, and E. Kominami, “Human light chain 3/MAP1LC3B is cleaved at its carboxyl-terminal Met(121) to expose Gly120 for lipidation and targeting to autophagosomal membranes,” J. Biol. Chem.279(46), 47704–47710 (2004).
[CrossRef] [PubMed]

N. Mizushima, “Methods for monitoring autophagy,” Int. J. Biochem. Cell Biol.36(12), 2491–2502 (2004).
[CrossRef] [PubMed]

2003 (2)

H. H. Cui, J. G. Valdez, J. A. Steinkamp, and H. A. Crissman, “Fluorescence lifetime-based discrimination and quantification of cellular DNA and RNA with phase-sensitive flow cytometry,” Cytometry A52(1), 46–55 (2003).
[CrossRef] [PubMed]

V. Calleja, S. M. Ameer-Beg, B. Vojnovic, R. Woscholski, J. Downward, and B. Larijani, “Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy,” Biochem. J.372(1), 33–40 (2003).
[CrossRef] [PubMed]

2002 (2)

K. Suhling, J. Siegel, D. Phillips, P. M. French, S. Lévêque-Fort, S. E. Webb, and D. M. Davis, “Imaging the environment of green fluorescent protein,” Biophys. J.83(6), 3589–3595 (2002).
[CrossRef] [PubMed]

J. Liang, J. Zubovitz, T. Petrocelli, R. Kotchetkov, M. K. Connor, K. Han, J. H. Lee, S. Ciarallo, C. Catzavelos, R. Beniston, E. Franssen, and J. M. Slingerland, “PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest,” Nat. Med.8(10), 1153–1160 (2002).
[CrossRef] [PubMed]

2001 (1)

D. B. Munafó and M. I. Colombo, “A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation,” J. Cell Sci.114(Pt 20), 3619–3629 (2001).
[PubMed]

2000 (1)

Y. Kabeya, N. Mizushima, T. Ueno, A. Yamamoto, T. Kirisako, T. Noda, E. Kominami, Y. Ohsumi, and T. Yoshimori, “LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing,” EMBO J.19(21), 5720–5728 (2000).
[CrossRef] [PubMed]

1999 (2)

R. Pepperkok, A. Squire, S. Geley, and P. I. Bastiaens, “Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy,” Curr. Biol.9(5), 269–274 (1999).
[CrossRef] [PubMed]

J. A. Steinkamp and J. F. Keij, “Fluorescence intensity and lifetime measurement of free and particle-bound fluorophore in a sample stream by phase-sensitive flow cytometry,” Rev. Sci. Instrum.70(12), 4682–4688 (1999).
[CrossRef]

1994 (1)

J. A. Steinkamp, “Phase-sensitive detection methods for resolving fluorescence emission signals and directly quantifying lifetime,” Methods Cell Biol.42(Pt B), 627–640 (1994).
[CrossRef] [PubMed]

1993 (2)

B. G. Pinsky, J. J. Ladasky, J. R. Lakowicz, K. Berndt, and R. A. Hoffman, “Phase-resolved fluorescence lifetime measurements for flow cytometry,” Cytometry14(2), 123–135 (1993).
[CrossRef] [PubMed]

J. A. Steinkamp and H. A. Crissman, “Resolution of fluorescence signals from cells labeled with fluorochromes having different lifetimes by phase-sensitive flow cytometry,” Cytometry14(2), 210–216 (1993).
[CrossRef] [PubMed]

Ameer-Beg, S. M.

V. Calleja, S. M. Ameer-Beg, B. Vojnovic, R. Woscholski, J. Downward, and B. Larijani, “Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy,” Biochem. J.372(1), 33–40 (2003).
[CrossRef] [PubMed]

Andersen, J. S.

A. R. Kristensen, S. Schandorff, M. Høyer-Hansen, M. O. Nielsen, M. Jäättelä, J. Dengjel, and J. S. Andersen, “Ordered organelle degradation during starvation-induced autophagy,” Mol. Cell. Proteomics7(12), 2419–2428 (2008).
[CrossRef] [PubMed]

Aoki, Y.

T. Nakabayashi, I. Nagao, M. Kinjo, Y. Aoki, M. Tanaka, and N. Ohta, “Stress-induced environmental changes in a single cell as revealed by fluorescence lifetime imaging,” Photochem. Photobiol. Sci.7(6), 671–674 (2008).
[CrossRef] [PubMed]

Bampton, E. T. W.

E. T. W. Bampton, C. G. Goemans, D. Niranjan, N. Mizushima, and A. M. Tolkovsky, “The dynamics of autophagy visualized in live cells - From autophagosome formation to fusion with endo/lysosomes,” Autophagy1(1), 23–37 (2005).
[CrossRef] [PubMed]

Barteneva, N. S.

N. S. Barteneva, E. Fasler-Kan, and I. A. Vorobjev, “Imaging flow cytometry: coping with heterogeneity in biological systems,” J. Histochem. Cytochem.60(10), 723–733 (2012).
[PubMed]

Bastiaens, P. I.

R. Pepperkok, A. Squire, S. Geley, and P. I. Bastiaens, “Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy,” Curr. Biol.9(5), 269–274 (1999).
[CrossRef] [PubMed]

Beniston, R.

J. Liang, J. Zubovitz, T. Petrocelli, R. Kotchetkov, M. K. Connor, K. Han, J. H. Lee, S. Ciarallo, C. Catzavelos, R. Beniston, E. Franssen, and J. M. Slingerland, “PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest,” Nat. Med.8(10), 1153–1160 (2002).
[CrossRef] [PubMed]

Berndt, K.

B. G. Pinsky, J. J. Ladasky, J. R. Lakowicz, K. Berndt, and R. A. Hoffman, “Phase-resolved fluorescence lifetime measurements for flow cytometry,” Cytometry14(2), 123–135 (1993).
[CrossRef] [PubMed]

Brady, N. R.

P. Hundeshagen, A. Hamacher-Brady, R. Eils, and N. R. Brady, “Concurrent detection of autolysosome formation and lysosomal degradation by flow cytometry in a high-content screen for inducers of autophagy,” BMC Biol.9(1), 38 (2011).
[CrossRef] [PubMed]

Caesar, K.

K. Elgass, K. Caesar, F. Schleifenbaum, Y. D. Stierhof, A. J. Meixner, and K. Harter, “Novel application of fluorescence lifetime and fluorescence microscopy enables quantitative access to subcellular dynamics in plant cells,” PLoS ONE4(5), e5716 (2009).
[CrossRef] [PubMed]

Calleja, V.

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M. Zhao, P. G. Schiro, J. S. Kuo, K. M. Koehler, D. E. Sabath, V. Popov, Q. Feng, and D. T. Chiu, “An automated high-throughput counting method for screening circulating tumor cells in peripheral blood,” Anal. Chem.85(4), 2465–2471 (2013).
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I. M. Chu, L. Hengst, and J. M. Slingerland, “The Cdk inhibitor p27 in human cancer: prognostic potential and relevance to anticancer therapy,” Nat. Rev. Cancer8(4), 253–267 (2008).
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J. Liang, J. Zubovitz, T. Petrocelli, R. Kotchetkov, M. K. Connor, K. Han, J. H. Lee, S. Ciarallo, C. Catzavelos, R. Beniston, E. Franssen, and J. M. Slingerland, “PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest,” Nat. Med.8(10), 1153–1160 (2002).
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A. H. Clayton, Q. S. Hanley, and P. J. Verveer, “Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data,” J. Microsc.213(1), 1–5 (2004).
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J. Liang, J. Zubovitz, T. Petrocelli, R. Kotchetkov, M. K. Connor, K. Han, J. H. Lee, S. Ciarallo, C. Catzavelos, R. Beniston, E. Franssen, and J. M. Slingerland, “PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest,” Nat. Med.8(10), 1153–1160 (2002).
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A. R. Kristensen, S. Schandorff, M. Høyer-Hansen, M. O. Nielsen, M. Jäättelä, J. Dengjel, and J. S. Andersen, “Ordered organelle degradation during starvation-induced autophagy,” Mol. Cell. Proteomics7(12), 2419–2428 (2008).
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B. Seefeldt, R. Kasper, T. Seidel, P. Tinnefeld, K. J. Dietz, M. Heilemann, and M. Sauer, “Fluorescent proteins for single-molecule fluorescence applications,” J Biophotonics1(1), 74–82 (2008).
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V. Calleja, S. M. Ameer-Beg, B. Vojnovic, R. Woscholski, J. Downward, and B. Larijani, “Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy,” Biochem. J.372(1), 33–40 (2003).
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M. A. Naivar, J. D. Parson, M. E. Wilder, R. C. Habbersett, B. S. Edwards, L. Sklar, J. P. Nolan, S. W. Graves, J. C. Martin, J. H. Jett, and J. P. Freyer, “Open, reconfigurable cytometric acquisition system: ORCAS,” Cytometry A71(11), 915–924 (2007).
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P. Hundeshagen, A. Hamacher-Brady, R. Eils, and N. R. Brady, “Concurrent detection of autolysosome formation and lysosomal degradation by flow cytometry in a high-content screen for inducers of autophagy,” BMC Biol.9(1), 38 (2011).
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J. Liang, J. Zubovitz, T. Petrocelli, R. Kotchetkov, M. K. Connor, K. Han, J. H. Lee, S. Ciarallo, C. Catzavelos, R. Beniston, E. Franssen, and J. M. Slingerland, “PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest,” Nat. Med.8(10), 1153–1160 (2002).
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K. Suhling, J. Siegel, D. Phillips, P. M. French, S. Lévêque-Fort, S. E. Webb, and D. M. Davis, “Imaging the environment of green fluorescent protein,” Biophys. J.83(6), 3589–3595 (2002).
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M. A. Naivar, J. D. Parson, M. E. Wilder, R. C. Habbersett, B. S. Edwards, L. Sklar, J. P. Nolan, S. W. Graves, J. C. Martin, J. H. Jett, and J. P. Freyer, “Open, reconfigurable cytometric acquisition system: ORCAS,” Cytometry A71(11), 915–924 (2007).
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A. Esposito, H. C. Gerritsen, and F. S. Wouters, “Fluorescence lifetime heterogeneity resolution in the frequency domain by lifetime moments analysis,” Biophys. J.89(6), 4286–4299 (2005).
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E. T. W. Bampton, C. G. Goemans, D. Niranjan, N. Mizushima, and A. M. Tolkovsky, “The dynamics of autophagy visualized in live cells - From autophagosome formation to fusion with endo/lysosomes,” Autophagy1(1), 23–37 (2005).
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P. Hundeshagen, A. Hamacher-Brady, R. Eils, and N. R. Brady, “Concurrent detection of autolysosome formation and lysosomal degradation by flow cytometry in a high-content screen for inducers of autophagy,” BMC Biol.9(1), 38 (2011).
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I. Tanida, T. Yamaji, T. Ueno, S. Ishiura, E. Kominami, and K. Hanada, “Consideration about negative controls for LC3 and expression vectors for four colored fluorescent protein-LC3 negative controls,” Autophagy4(1), 131–134 (2008).
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A. H. Clayton, Q. S. Hanley, and P. J. Verveer, “Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data,” J. Microsc.213(1), 1–5 (2004).
[CrossRef] [PubMed]

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K. Elgass, K. Caesar, F. Schleifenbaum, Y. D. Stierhof, A. J. Meixner, and K. Harter, “Novel application of fluorescence lifetime and fluorescence microscopy enables quantitative access to subcellular dynamics in plant cells,” PLoS ONE4(5), e5716 (2009).
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B. Seefeldt, R. Kasper, T. Seidel, P. Tinnefeld, K. J. Dietz, M. Heilemann, and M. Sauer, “Fluorescent proteins for single-molecule fluorescence applications,” J Biophotonics1(1), 74–82 (2008).
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I. M. Chu, L. Hengst, and J. M. Slingerland, “The Cdk inhibitor p27 in human cancer: prognostic potential and relevance to anticancer therapy,” Nat. Rev. Cancer8(4), 253–267 (2008).
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B. G. Pinsky, J. J. Ladasky, J. R. Lakowicz, K. Berndt, and R. A. Hoffman, “Phase-resolved fluorescence lifetime measurements for flow cytometry,” Cytometry14(2), 123–135 (1993).
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R. Cao, V. Pankayatselvan, and J. P. Houston, “Cytometric sorting based on the fluorescence lifetime of spectrally overlapping signals,” Opt. Express21(12), 14816–14831 (2013).
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J. P. Houston, M. A. Naivar, and J. P. Freyer, “Capture of fluorescence decay times by flow cytometry,” Curr. Protoc. Cytom. (2012).
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J. P. Houston, M. A. Naivar, P. Jenkins, and J. P. Freyer, “Digital Analysis and Sorting of Fluorescence Lifetime by Flow Cytometry,” Cytometry A77(9), 861–872 (2010).
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A. R. Kristensen, S. Schandorff, M. Høyer-Hansen, M. O. Nielsen, M. Jäättelä, J. Dengjel, and J. S. Andersen, “Ordered organelle degradation during starvation-induced autophagy,” Mol. Cell. Proteomics7(12), 2419–2428 (2008).
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P. Hundeshagen, A. Hamacher-Brady, R. Eils, and N. R. Brady, “Concurrent detection of autolysosome formation and lysosomal degradation by flow cytometry in a high-content screen for inducers of autophagy,” BMC Biol.9(1), 38 (2011).
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M. C. Hung and W. Link, “Protein localization in disease and therapy,” J. Cell Sci.124(20), 3381–3392 (2011).
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I. Tanida, T. Yamaji, T. Ueno, S. Ishiura, E. Kominami, and K. Hanada, “Consideration about negative controls for LC3 and expression vectors for four colored fluorescent protein-LC3 negative controls,” Autophagy4(1), 131–134 (2008).
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E. A. Corcelle, P. Puustinen, and M. Jäättelä, “Apoptosis and autophagy: Targeting autophagy signalling in cancer cells -‘trick or treats’?” FEBS J.276(21), 6084–6096 (2009).
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A. R. Kristensen, S. Schandorff, M. Høyer-Hansen, M. O. Nielsen, M. Jäättelä, J. Dengjel, and J. S. Andersen, “Ordered organelle degradation during starvation-induced autophagy,” Mol. Cell. Proteomics7(12), 2419–2428 (2008).
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J. P. Houston, M. A. Naivar, P. Jenkins, and J. P. Freyer, “Digital Analysis and Sorting of Fluorescence Lifetime by Flow Cytometry,” Cytometry A77(9), 861–872 (2010).
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M. A. Naivar, J. D. Parson, M. E. Wilder, R. C. Habbersett, B. S. Edwards, L. Sklar, J. P. Nolan, S. W. Graves, J. C. Martin, J. H. Jett, and J. P. Freyer, “Open, reconfigurable cytometric acquisition system: ORCAS,” Cytometry A71(11), 915–924 (2007).
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B. Seefeldt, R. Kasper, T. Seidel, P. Tinnefeld, K. J. Dietz, M. Heilemann, and M. Sauer, “Fluorescent proteins for single-molecule fluorescence applications,” J Biophotonics1(1), 74–82 (2008).
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J. A. Steinkamp and J. F. Keij, “Fluorescence intensity and lifetime measurement of free and particle-bound fluorophore in a sample stream by phase-sensitive flow cytometry,” Rev. Sci. Instrum.70(12), 4682–4688 (1999).
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S. Kimura, T. Noda, and T. Yoshimori, “Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3,” Autophagy3(5), 452–460 (2007).
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T. Ito, S. Oshita, T. Nakabayashi, F. Sun, M. Kinjo, and N. Ohta, “Fluorescence lifetime images of green fluorescent protein in HeLa cells during TNF-alpha induced apoptosis,” Photochem. Photobiol. Sci.8(6), 763–767 (2009).
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T. Nakabayashi, H. P. Wang, M. Kinjo, and N. Ohta, “Application of fluorescence lifetime imaging of enhanced green fluorescent protein to intracellular pH measurements,” Photochem. Photobiol. Sci.7(6), 668–670 (2008).
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T. Nakabayashi, I. Nagao, M. Kinjo, Y. Aoki, M. Tanaka, and N. Ohta, “Stress-induced environmental changes in a single cell as revealed by fluorescence lifetime imaging,” Photochem. Photobiol. Sci.7(6), 671–674 (2008).
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Y. Kabeya, N. Mizushima, T. Ueno, A. Yamamoto, T. Kirisako, T. Noda, E. Kominami, Y. Ohsumi, and T. Yoshimori, “LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing,” EMBO J.19(21), 5720–5728 (2000).
[CrossRef] [PubMed]

Koehler, K. M.

M. Zhao, P. G. Schiro, J. S. Kuo, K. M. Koehler, D. E. Sabath, V. Popov, Q. Feng, and D. T. Chiu, “An automated high-throughput counting method for screening circulating tumor cells in peripheral blood,” Anal. Chem.85(4), 2465–2471 (2013).
[CrossRef] [PubMed]

Kominami, E.

I. Tanida, T. Yamaji, T. Ueno, S. Ishiura, E. Kominami, and K. Hanada, “Consideration about negative controls for LC3 and expression vectors for four colored fluorescent protein-LC3 negative controls,” Autophagy4(1), 131–134 (2008).
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I. Tanida, T. Ueno, and E. Kominami, “Human light chain 3/MAP1LC3B is cleaved at its carboxyl-terminal Met(121) to expose Gly120 for lipidation and targeting to autophagosomal membranes,” J. Biol. Chem.279(46), 47704–47710 (2004).
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Y. Kabeya, N. Mizushima, T. Ueno, A. Yamamoto, T. Kirisako, T. Noda, E. Kominami, Y. Ohsumi, and T. Yoshimori, “LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing,” EMBO J.19(21), 5720–5728 (2000).
[CrossRef] [PubMed]

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J. Liang, J. Zubovitz, T. Petrocelli, R. Kotchetkov, M. K. Connor, K. Han, J. H. Lee, S. Ciarallo, C. Catzavelos, R. Beniston, E. Franssen, and J. M. Slingerland, “PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest,” Nat. Med.8(10), 1153–1160 (2002).
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A. R. Kristensen, S. Schandorff, M. Høyer-Hansen, M. O. Nielsen, M. Jäättelä, J. Dengjel, and J. S. Andersen, “Ordered organelle degradation during starvation-induced autophagy,” Mol. Cell. Proteomics7(12), 2419–2428 (2008).
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A. Kuma, M. Matsui, and N. Mizushima, “LC3, an autophagosome marker, can be incorporated into protein aggregates independent of autophagy: caution in the interpretation of LC3 localization,” Autophagy3(4), 323–328 (2007).
[PubMed]

Kuo, J. S.

M. Zhao, P. G. Schiro, J. S. Kuo, K. M. Koehler, D. E. Sabath, V. Popov, Q. Feng, and D. T. Chiu, “An automated high-throughput counting method for screening circulating tumor cells in peripheral blood,” Anal. Chem.85(4), 2465–2471 (2013).
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Ladasky, J. J.

B. G. Pinsky, J. J. Ladasky, J. R. Lakowicz, K. Berndt, and R. A. Hoffman, “Phase-resolved fluorescence lifetime measurements for flow cytometry,” Cytometry14(2), 123–135 (1993).
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B. G. Pinsky, J. J. Ladasky, J. R. Lakowicz, K. Berndt, and R. A. Hoffman, “Phase-resolved fluorescence lifetime measurements for flow cytometry,” Cytometry14(2), 123–135 (1993).
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B. Treanor, P. M. Lanigan, K. Suhling, T. Schreiber, I. Munro, M. A. Neil, D. Phillips, D. M. Davis, and P. M. French, “Imaging fluorescence lifetime heterogeneity applied to GFP-tagged MHC protein at an immunological synapse,” J. Microsc.217(1), 36–43 (2005).
[CrossRef] [PubMed]

Larijani, B.

V. Calleja, S. M. Ameer-Beg, B. Vojnovic, R. Woscholski, J. Downward, and B. Larijani, “Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy,” Biochem. J.372(1), 33–40 (2003).
[CrossRef] [PubMed]

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J. Liang, J. Zubovitz, T. Petrocelli, R. Kotchetkov, M. K. Connor, K. Han, J. H. Lee, S. Ciarallo, C. Catzavelos, R. Beniston, E. Franssen, and J. M. Slingerland, “PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest,” Nat. Med.8(10), 1153–1160 (2002).
[CrossRef] [PubMed]

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K. Suhling, J. Siegel, D. Phillips, P. M. French, S. Lévêque-Fort, S. E. Webb, and D. M. Davis, “Imaging the environment of green fluorescent protein,” Biophys. J.83(6), 3589–3595 (2002).
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Levine, B.

N. Mizushima, T. Yoshimori, and B. Levine, “Methods in Mammalian Autophagy Research,” Cell140(3), 313–326 (2010).
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F. Cecconi and B. Levine, “The role of autophagy in mammalian development: cell makeover rather than cell death,” Dev. Cell15(3), 344–357 (2008).
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J. Liang, J. Zubovitz, T. Petrocelli, R. Kotchetkov, M. K. Connor, K. Han, J. H. Lee, S. Ciarallo, C. Catzavelos, R. Beniston, E. Franssen, and J. M. Slingerland, “PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest,” Nat. Med.8(10), 1153–1160 (2002).
[CrossRef] [PubMed]

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M. C. Hung and W. Link, “Protein localization in disease and therapy,” J. Cell Sci.124(20), 3381–3392 (2011).
[CrossRef] [PubMed]

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M. A. Naivar, J. D. Parson, M. E. Wilder, R. C. Habbersett, B. S. Edwards, L. Sklar, J. P. Nolan, S. W. Graves, J. C. Martin, J. H. Jett, and J. P. Freyer, “Open, reconfigurable cytometric acquisition system: ORCAS,” Cytometry A71(11), 915–924 (2007).
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R. Mathew and E. White, “Autophagy in tumorigenesis and energy metabolism: friend by day, foe by night,” Curr. Opin. Genet. Dev.21(1), 113–119 (2011).
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A. Kuma, M. Matsui, and N. Mizushima, “LC3, an autophagosome marker, can be incorporated into protein aggregates independent of autophagy: caution in the interpretation of LC3 localization,” Autophagy3(4), 323–328 (2007).
[PubMed]

Meixner, A. J.

K. Elgass, K. Caesar, F. Schleifenbaum, Y. D. Stierhof, A. J. Meixner, and K. Harter, “Novel application of fluorescence lifetime and fluorescence microscopy enables quantitative access to subcellular dynamics in plant cells,” PLoS ONE4(5), e5716 (2009).
[CrossRef] [PubMed]

Mizushima, N.

N. Mizushima, T. Yoshimori, and B. Levine, “Methods in Mammalian Autophagy Research,” Cell140(3), 313–326 (2010).
[CrossRef] [PubMed]

A. Kuma, M. Matsui, and N. Mizushima, “LC3, an autophagosome marker, can be incorporated into protein aggregates independent of autophagy: caution in the interpretation of LC3 localization,” Autophagy3(4), 323–328 (2007).
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E. T. W. Bampton, C. G. Goemans, D. Niranjan, N. Mizushima, and A. M. Tolkovsky, “The dynamics of autophagy visualized in live cells - From autophagosome formation to fusion with endo/lysosomes,” Autophagy1(1), 23–37 (2005).
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A. Pliss, L. Zhao, T. Y. Ohulchanskyy, J. Qu, and P. N. Prasad, “Fluorescence lifetime of fluorescent proteins as an intracellular environment probe sensing the cell cycle progression,” ACS Chem. Biol.7(8), 1385–1392 (2012).
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Zhao, M.

M. Zhao, P. G. Schiro, J. S. Kuo, K. M. Koehler, D. E. Sabath, V. Popov, Q. Feng, and D. T. Chiu, “An automated high-throughput counting method for screening circulating tumor cells in peripheral blood,” Anal. Chem.85(4), 2465–2471 (2013).
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Zubovitz, J.

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ACS Chem. Biol. (1)

A. Pliss, L. Zhao, T. Y. Ohulchanskyy, J. Qu, and P. N. Prasad, “Fluorescence lifetime of fluorescent proteins as an intracellular environment probe sensing the cell cycle progression,” ACS Chem. Biol.7(8), 1385–1392 (2012).
[CrossRef] [PubMed]

Anal. Chem. (1)

M. Zhao, P. G. Schiro, J. S. Kuo, K. M. Koehler, D. E. Sabath, V. Popov, Q. Feng, and D. T. Chiu, “An automated high-throughput counting method for screening circulating tumor cells in peripheral blood,” Anal. Chem.85(4), 2465–2471 (2013).
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Figures (3)

Fig. 1
Fig. 1

Schematic of the time-resolved flow cytometry (TRFC) system. A modulated laser excites individual cells that are flowing in single file (direction is out of the page in the image). Fluorescence is collected (side scatter also, but not illustrated for brevity) whereby the PMT measures the phase-shifted frequency modulated signal. For fluorescence lifetime acquisition analog mixing hardware permit the measurement of the tangent of the phase-shift (proportional to lifetime), which is collected by a high-speed data acquisition system.

Fig. 2
Fig. 2

Detection of EGFP in MCF-7 breast cancer cells expressing EGFP, EGFP-LC3, or EGFP-ΔLC3 under non-starved conditions (a)-(c), and after autophagy induction by amino acid starvation for 3 hours (d)-(f). MCF-7 cells expressing EGFP (a) and (d), EGFP-LC3 (b) and (e), or EGFP-ΔLC3 (c) and (f) were imaged with confocal microscopy to show fluorescence intensity detected between 500 and 560 nm. These data are representative of at least 3 independent experiments.

Fig. 3
Fig. 3

Decreased mean fluorescence lifetime (MFL) of EGFP upon subcellular localization to autophagosomes. Histograms indicating the mean fluorescence intensity (MFI) of amino acid-starved MCF-7 cells expressing EGFP (a), EGFP-LC3 (b), or EGFP-DLC3 (D = Δ) (c). MFI is shown for MCF-7 cells not transfected with EGFP as a negative control. Histograms indicating MFL in amino acid-starved MCF-7 cells expressing EGFP (d), EGFP-LC3 (e), or EGFP-DLC3 (D = Δ) (f). MFI data were obtained by 488 nm excitation and 530/30 nm emission using the BD Accuri C6 Flow Cytometer and the MFL data were obtained by 488 nm excitation and > 490-nm emission on the TRFC instrument described previously. These data are representative of 5 independent experiments.

Equations (2)

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τ= tan(ϕ) ω
F(t)=a(1+m i=1 N cos((2i1)ωt ϕ i ) 2i1 ) e b (t t 0 ) 2

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