Phase Differential Enhancement of FLIM to Distinguish FRET Components of a Biosensor for Monitoring Molecular Activity of Membrane Type 1 Matrix Metalloproteinase in Live Cells

Fluorescence lifetime-resolved imaging microscopy (FLIM) has been used to monitor the enzymatic activity of a proteolytic enzyme, Membrane Type 1 Matrix Metalloproteinase (MT1-MMP), with a recently developed FRET-based biosensor in vitro and in live HeLa and HT1080 cells. MT1-MMP is a collagenaise that is involved in the destruction of extra-cellular matrix (ECM) proteins, as well as in various cellular functions including migration. The increased expression of MT1-MMP has been positively correlated with the invasive potential of tumor cells. However, the precise spatiotemporal activation patterns of MT1-MMP in live cells are still not well-established. The activity of MT1-MMP was examined with our biosensor in live cells. Imaging of live cells was performed with full-field frequency-domain FLIM. Image analysis was carried out both with polar plots and phase differential enhancement. Phase differential enhancement, which is similar to phase suppression, is shown to facilitate the differentiation between different conformations of the MT1-MMP biosensor in live cells when the lifetime differences are small. FLIM carried out in differential enhancement or phase suppression modes, requires only two acquired phase images, and permits rapid imaging of the activity of MT1-MMP in live cells.

[1]  E. Gratton,et al.  A continuously variable frequency cross-correlation phase fluorometer with picosecond resolution. , 1983, Biophysical journal.

[2]  R. Clegg,et al.  Fluorescence Lifetime-Resolved Imaging Microscopy (FLIM) , 1996 .

[3]  N. Otsu.  A threshold selection method from gray level histograms , 1979 .

[4]  Elliot L. Botvinick,et al.  Visualizing the mechanical activation of Src , 2005, Nature.

[5]  H. Mori,et al.  CD44 directs membrane‐type 1 matrix metalloproteinase to lamellipodia by associating with its hemopexin‐like domain , 2002, The EMBO journal.

[6]  Ammasi Periasamy,et al.  Characterization of an improved donor fluorescent protein for Forster resonance energy transfer microscopy. , 2008, Journal of biomedical optics.

[7]  Th. Förster.  Zwischenmolekulare Energiewanderung und Fluoreszenz , 1948 .

[8]  R. Tsien,et al.  Monitoring protein conformations and interactions by fluorescence resonance energy transfer between mutants of green fluorescent protein. , 2000, Methods in enzymology.

[9]  Raymond F. Chen,et al.  Biochemical fluorescence : concepts , 1975 .

[10]  L. Matrisian,et al.  Matrix metalloproteinases in tumor-host cell communication. , 2002, Differentiation; research in biological diversity.

[11]  Robert M. Clegg,et al.  Flim Microscopy in Biology and Medicine , 2009 .

[12]  Robert M. Clegg,et al.  Fluorescence lifetime imaging microscopy (FLIM): Spatial resolution of microstructures on the nanosecond time scale , 1993 .

[13]  Z. Werb,et al.  How matrix metalloproteinases regulate cell behavior. , 2001, Annual review of cell and developmental biology.

[14]  A. Hoppe,et al.  Polarized fluorescence resonance energy transfer microscopy. , 2004, Biophysical journal.

[15]  Robert M. Clegg,et al.  Rapid acquisition, analysis, and display of fluorescence lifetime-resolved images for real-time applications , 1997 .

[16]  遠藤 一平.  Cleavage of syndecan-1 by membrane type matrix metalloproteinase-1 stimulates cell migration , 2003 .

[17]  R. Clegg,et al.  Polar Plot Representation for Frequency-Domain Analysis of Fluorescence Lifetimes , 2005, Journal of Fluorescence.

[18]  A. Visser,et al.  Effects of Refractive Index and Viscosity on Fluorescence and Anisotropy Decays of Enhanced Cyan and Yellow Fluorescent Proteins , 2005, Journal of Fluorescence.

[19]  Patrick S Daugherty,et al.  Evolutionary optimization of fluorescent proteins for intracellular FRET , 2005, Nature Biotechnology.

[20]  Thomas M. Jovin,et al.  Time-resolved imaging fluorescence microscopy , 1992, Photonics West - Lasers and Applications in Science and Engineering.

[21]  D. Gingras,et al.  Src-dependent Phosphorylation of Membrane Type I Matrix Metalloproteinase on Cytoplasmic Tyrosine 573 , 2007, Journal of Biological Chemistry.

[22]  G. Weber,et al.  Resolution of the fluorescence lifetimes in a heterogeneous system by phase and modulation measurements , 1981 .

[23]  B. Spring,et al.  Image analysis for denoising full‐field frequency‐domain fluorescence lifetime images , 2009, Journal of microscopy.

[24]  R. D. Spencer,et al.  MEASUREMENTS OF SUBNANOSECOND FLUORESCENCE LIFETIMES WITH A CROSS‐CORRELATION PHASE FLUOROMETER * , 1969 .

[25]  E. Gratton,et al.  The phasor approach to fluorescence lifetime imaging analysis. , 2008, Biophysical journal.

[26]  J. Lakowicz,et al.  Phase-sensitive fluorescence spectroscopy: a new method to resolve fluorescence lifetimes or emission spectra of components in a mixture of fluorophores. , 1981, Journal of biochemical and biophysical methods.

[27]  R. Nagle,et al.  Membrane type-1-matrix metalloproteinase expressed by prostate carcinoma cells cleaves human laminin-5 beta3 chain and induces cell migration. , 2003, Cancer research.

[28]  L. McGown,et al.  Phase-Resolved Fluorescence in Chemical Analysis , 1987 .

[29]  J. Lakowicz,et al.  Fluorescence lifetime imaging of free and protein-bound NADH. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[30]  R. Tsien,et al.  Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein , 2004, Nature Biotechnology.

[31]  E. Gratton,et al.  The Measurement and Analysis of Heterogeneous Emissions by Multifrequency Phase and Modulation Fluorometry , 1984 .

[32]  Jing Xu,et al.  Visualization of Polarized Membrane Type 1 Matrix Metalloproteinase Activity in Live Cells by Fluorescence Resonance Energy Transfer Imaging* , 2008, Journal of Biological Chemistry.

[33]  J. Lakowicz.  Principles of fluorescence spectroscopy , 1983 .

[34]  Alex Y Strongin,et al.  Simultaneous visualization of protumorigenic Src and MT1-MMP activities with fluorescence resonance energy transfer. , 2010, Cancer research.

[35]  Thomas M. Jovin,et al.  Lifetime-resolved fluorescence imaging , 1994, Photonics West - Lasers and Applications in Science and Engineering.

[36]  R. Young,et al.  Quantitation of fluorescence energy transfer between cell surface proteins via fluorescence donor photobleaching kinetics. , 1994, Biophysical journal.

[37]  Walter Kolch,et al.  High-precision FLIM-FRET in fixed and living cells reveals heterogeneity in a simple CFP-YFP fusion protein. , 2007, Biophysical chemistry.

[38]  Pascal Pernot,et al.  Complex fluorescence of the cyan fluorescent protein: comparisons with the H148D variant and consequences for quantitative cell imaging. , 2008, Biochemistry.

[39]  R. Hartig,et al.  Photophysics of Clomeleon by FLIM: discriminating excited state reactions along neuronal development. , 2007, Biophysical journal.

[40]  J. Guan,et al.  FAK-mediated src phosphorylation of endophilin A2 inhibits endocytosis of MT1-MMP and promotes ECM degradation. , 2005, Developmental cell.

[41]  Mark A Rizzo,et al.  An improved cyan fluorescent protein variant useful for FRET , 2004, Nature Biotechnology.

[42]  J. Lakowicz,et al.  Analysis of excited-state processes by phase-modulation fluorescence spectroscopy. , 1982, Biophysical chemistry.

[43]  Govindjee,et al.  Fluorescence Lifetime Imaging (FLI) in Real-Time - a New Technique in Photosynthesis Research , 2000, Photosynthetica.

[44]  Joachim Goedhart,et al.  Quantitative lifetime unmixing of multiexponentially decaying fluorophores using single-frequency fluorescence lifetime imaging microscopy. , 2008, Biophysical journal.

[45]  Philippe Chavrier,et al.  Matrix invasion by tumour cells: a focus on MT1-MMP trafficking to invadopodia , 2009, Journal of Cell Science.

[46]  Motoharu Seiki,et al.  Membrane-type 1 matrix metalloproteinase: a key enzyme for tumor invasion. , 2003, Cancer letters.

[47]  P. Verveer,et al.  Graphical representation and multicomponent analysis of single‐frequency fluorescence lifetime imaging microscopy data , 2004, Journal of microscopy.

[48]  J. Lakowicz,et al.  Resolution of Heterogeneous Fluorescence by Phase-sensitive Fluorescence Spectroscopy. , 1982, Biophysical journal.

[49]  I Gryczynski,et al.  Background suppression in frequency-domain fluorometry. , 2000, Analytical biochemistry.

[50]  Robert M. Clegg,et al.  General concerns of FLIM data representation and analysis: Frequency-domain model-free analysis , 2009 .

[51]  J. Lakowicz,et al.  Analysis of fluorescence decay kinetics from variable-frequency phase shift and modulation data. , 1984, Biophysical journal.

[52]  Hiroshi Ohno,et al.  Cytoplasmic tail–dependent internalization of membrane-type 1 matrix metalloproteinase is important for its invasion-promoting activity , 2001, The Journal of cell biology.

[53]  Motoharu Seiki,et al.  MT1‐MMP: A potent modifier of pericellular microenvironment , 2006, Journal of cellular physiology.

[54]  E Gratton,et al.  Multifrequency phase and modulation fluorometry. , 1984, Annual review of biophysics and bioengineering.

[55]  Thomas M. Jovin,et al.  FRET Microscopy: Digital Imaging of Fluorescence Resonance Energy Transfer. Application in Cell Biology , 1989 .

[56]  B. Valeur,et al.  Molecular Fluorescence: Principles and Applications , 2001 .

[57]  Steven Shapiro,et al.  Tumor cell traffic through the extracellular matrix is controlled by the membrane-anchored collagenase MT1-MMP , 2004, The Journal of cell biology.

[58]  J. Lakowicz,et al.  Theory of phase-modulation fluorescence spectroscopy for excited-state processes. , 1982, Biophysical chemistry.

[59]  R. Tsien,et al.  Fluorescent indicators for Ca2+based on green fluorescent proteins and calmodulin , 1997, Nature.

[60]  Th. Förster.  Fluoreszenz organischer Verbindungen , 1951 .

[61]  R. Clegg.  Fluorescence resonance energy transfer and nucleic acids. , 1992, Methods in enzymology.

[62]  Michael Z. Lin,et al.  Improving the photostability of bright monomeric orange and red fluorescent proteins , 2008, Nature Methods.

[63]  H. Mori,et al.  Membrane-Type 1 Matrix Metalloproteinase Cleaves Cd44 and Promotes Cell Migration , 2001, The Journal of cell biology.

[64]  N. Chaffey.  Red fluorescent protein , 2001 .