title: Red-shifted fluorescent proteins monitor enzymatic activity in live HT-1080 cells with fluorescence lifetime imaging microscopy (FLIM).
abstract: Membrane type 1 matrix metalloproteinase (MT1-MMP) is a membrane-tethered collagenase primarily involved in the mechanical destruction of extracellular matrix proteins. MT1-MMP has also been shown to be upregulated in several types of cancers. Many coordinated functions of MT1-MMP during migration and invasion remain to be determined. In this paper, live cells from the invasive cell line HT-1080 were imaged using an intracellular Förster resonance energy transfer-based biosensor specific for MT1-MMP; a substrate specific for MT1-MMP was hybridized with the mOrange2 and mCherry fluorescent proteins to form the Förster resonance energy transfer-based sensor. The configuration of the biosensor was determined with fluorescence lifetime-resolved imaging microscopy using both a polar plot-based analysis and a rapid data acquisition modality of fluorescence lifetime-resolved imaging microscopy known as phase suppression. Both configurations of the biosensor (with or without cleavage by MT1-MMP) were clearly resolvable in the same cell. Changes in the configuration of the MT1-MMP biosensor were observed primarily along the edge of the cell following the removal of the MMP inhibitor GM6001. The intensities highlighted by phase suppression correlated well with the fractional intensities derived from the polar plot.
authors.affiliation: Center of Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: J P
authors.initials: JP
authors.lastname: Eichorst
authors.forename: R M
authors.initials: RM
authors.lastname: Clegg
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
mesh_terms: D015374:Biosensing Techniques / Q000379:methods; D045744:Cell Line, Tumor; D003584:Cytological Techniques / Q000379:methods*; D005347:Fibroblasts / Q000201:enzymology*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism*; D053511:Matrix Metalloproteinase 14 / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*
chemical_list: D008164:Luminescent Proteins; C507086:MMP14 protein, human; D053511:Matrix Metalloproteinase 14