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. Author manuscript; available in PMC: 2019 Apr 19.
Published in final edited form as: Cell Chem Biol. 2018 Jan 27;25(4):370–379.e4. doi: 10.1016/j.chembiol.2018.01.002

Figure 4. The ECFP/PEbody hybrid FRET biosensor for visualizing MT1-MMP activity on the surface of live cancer cells.

Figure 4

(A) The design strategy and the sensing mechanism of the biosensor. The biosensor was anchored on the extracellular surface using the transmembrane domain of PDGFR. The N-terminus of the transmembrane domain was linked to PEbody to allow for the R-PE binding. A MT1-MMP substrate sequence flanked by flexible linkers was inserted between ECFP and PEbody. Left: R-PE-staining of the intact biosensor allowed the energy transfer from ECFP to R-PE; Right: Following activation, MT1-MMP cleaved the biosensor substrate sequence, disrupted FRET, and reduced the FRET/R-PE ratio. (B) The DIC and FRET/R-PE ratio images before and after GM6001 inhibitor washout of the representative HeLa cells expressing the MT1-MMP ECFP/PEbody biosensor (i) with or (ii) without the full-length MT1-MMP gene. The color bar indicates the FRET/R-PE ratio, with hot and cold colors representing the high and low ratios, respectively. The white boxes indicate the region where the ratio was quantified for (C). Scale bar: 20 µm. (C) The average quantified time course of the normalized FRET/R-PE ratio from multiple HeLa cells in the GM6001 washout assay was compared in cells with and without the full-length MT1-MMP gene. Data are normalized with an individual basal FRET/R-PE ratio before GM6001 washout and represented as mean ± SD (n = 64 and 29; n is the number of analyzed cell contacts with and without the full-length MT1-MMP, respectively). See also Figure S3.