title: Genetically encoded FRET-based biosensor for imaging MMP-9 activity.
abstract: A genetically encoded Förster Resonance Energy Transfer (FRET)-based biosensor that continuously monitors matrix metalloproteinase 9 (MMP-9) activity was developed. MMP-9 is an extracellularly acting endopeptidase with a prominent role in development, learning and memory, cancer metastasis, and stroke. To assess the biological function of the protease, determining the precise kinetics and localization of MMP-9 activity is required. The nontoxic, genetically encoded FRET biosensor presented herein is anchored in the cellular membrane and thus provides an important advantage over currently employed probes. The biosensor allows the study of the proteolytic activity of MMP-9 with high temporal and subcellular resolution at the precise region of MMP-9 action on the cell. The applicability of the biosensor both in vitro and in living cells was demonstrated by ratiometrically analyzing the cleavage of the biosensor by a purified auto-activating mutant of MMP-9 and endogenously secreted protease in cultured tumor and neuronal cells. The precise kinetics of endogenous MMP-9 activity was measured, which demonstrates in a straight-forward manner the applicability of the biosensor concept.
authors.affiliation: Department of Molecular and Cellular Neurobiology, The Nencki Institute of Experimental Biology, ul. Ludwika Pasteura 3, Warszawa 02-093, Poland.
authors.forename: Michal
authors.initials: M
authors.lastname: Stawarski
authors.affiliation: Department of Molecular and Cellular Neurobiology, The Nencki Institute of Experimental Biology, ul. Ludwika Pasteura 3, Warszawa 02-093, Poland.
authors.forename: Izabela
authors.initials: I
authors.lastname: Rutkowska-Wlodarczyk
authors.affiliation: Department of Cellular Neurophysiology, Hannover Medical School, Carl-Neuberg-Strasse 1, Hannover 30625, Germany.
authors.forename: André
authors.initials: A
authors.lastname: Zeug
authors.affiliation: Department of Molecular and Cellular Neurobiology, The Nencki Institute of Experimental Biology, ul. Ludwika Pasteura 3, Warszawa 02-093, Poland.
authors.forename: Monika
authors.initials: M
authors.lastname: Bijata
authors.affiliation: Department of Biophysics, Warsaw University of Life Sciences, ul. Nowoursynowska 166, Warszawa 02-787, Poland; Department of Molecular and Cellular Neurobiology, The Nencki Institute of Experimental Biology, ul. Ludwika Pasteura 3, Warszawa 02-093, Poland.
authors.forename: Hubert
authors.initials: H
authors.lastname: Madej
authors.affiliation: Department of Molecular and Cellular Neurobiology, The Nencki Institute of Experimental Biology, ul. Ludwika Pasteura 3, Warszawa 02-093, Poland.
authors.forename: Leszek
authors.initials: L
authors.lastname: Kaczmarek
authors.affiliation: Department of Molecular and Cellular Neurobiology, The Nencki Institute of Experimental Biology, ul. Ludwika Pasteura 3, Warszawa 02-093, Poland. Electronic address: j.wlodarczyk@nencki.gov.pl.
authors.forename: Jakub
authors.initials: J
authors.lastname: Wlodarczyk
mesh_terms: D001483:Base Sequence; D015374:Biosensing Techniques; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism*; D059748:Proteolysis
chemical_list: D017931:DNA Primers; D020780:Matrix Metalloproteinase 9
keywords: Biosensors; Cancer-related processes; Förster resonance energy transfer (FRET); Long-term memory; Matrix metalloproteinase 9; Quantitative microscopy