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. 2012 Oct;248(1):77-89.
doi: 10.1111/j.1365-2818.2012.03652.x.

Red-shifted fluorescent proteins monitor enzymatic activity in live HT-1080 cells with fluorescence lifetime imaging microscopy (FLIM)

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Red-shifted fluorescent proteins monitor enzymatic activity in live HT-1080 cells with fluorescence lifetime imaging microscopy (FLIM)

J P Eichorst et al. J Microsc. 2012 Oct.

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.

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Figures

Fig. 1
Fig. 1
Time-resolved in vitro measurements of a purified solution of the MT1-MMP biosensor: (A) The polar coordinates of the MT1-MMP biosensor were measured before and after the biosensor was cleaved by trypsin. Measurements were taken at a modulation frequency of 40 MHz through the emission filter shown in (C). (B) The phase shift and demodulation of the mOrange2 was also measured on both configurations of the biosensor at an emission wavelength of 570 ± 10 nm. When measured and plotted on the polar plot at a modulation frequency of 40 MHz, there is a definite movement of the polar coordinate toward longer lifetimes after cleavage of the biosensor. (C) The normalized emission spectra of mOrange2 and mCherry are displayed along with the filter pass of the emission filter highlighted in (A) (and used for the live cell measurements). (D) Separate protein solutions of the intact biosensor and the biosensor cleaved by trypsin were examined in a polyacrylamide gel. The leftmost lane contains the intact biosensor and the centre lane contains the cleaved biosensor. The far right lane is the indicator lane displaying the reference masses.
Fig. 2
Fig. 2
Steady-state measurements taken on a purified solution of the MT1-MMP biosensor: (A) The emission spectrum of the intact MT1-MMP biosensor was measured with an excitation wavelength of 532 nm. Spectral unmixing was applied to resolve the contributions of the mOrange2 and mCherry separately. (B) The emission spectrum of the cleaved biosensor was also measured at an excitation wavelength of 532 nm. After the biosensor was cleaved, there is a definite loss in emission from the mCherry and an increase in fluorescence emission from the mOrange2 relative to (A) indicating a loss of FRET.
Fig. 3
Fig. 3
HT-1080 cell treated with the GM6001 inhibitor: (A) An intensity image of an HT-1080 cell taken during a homodyning FLIM experiment is presented with four regions of interest highlighted in white squares. (B) An IMD (intensity-masked display) image is shown where the red shading indicates where the fractional intensity of the intact biosensor is dominant relative to the cleaved biosensor. (C) In this IMD image, the areas in the cell where the fractional intensity from the cleaved biosensor (mOrange2) is dominant are shown in green. (D) When both (B) and (C) are combined, the resulting cell image shows that most of the collected intensity is being emitted from the intact biosensor as indicated by the red shading. (E) The pixels from the regions in interest in (A) are projected on the polar plot in these scatter plots. The plots indicate that there is likely a single lifetime pool near 2 ns representing the intact biosensor.
Fig. 4
Fig. 4
HT-1080 cell not treated with the GM6001 inhibitor: (A)Anintensity image of anHT-1080is shown with regions of interests (#1–4) highlighted. (B) Red shading is presented indicating where the fractional intensity from the intact biosensor is most prevalent. (C) Highlighted in green are the areas in the cell where the fractional intensity of the cleaved biosensor (mOrange2) is dominant in the image. As shown, these areas reside mostly on the edge of the cell. (D) Images (B) and (C) were then combined to form an image where the dominant fractional intensities of both configurations of the biosensor are highlighted together. (E) These scatter plots are projections of the pixels from the regions of interest in (A) on the polar plot. Two lifetime pools between 2 ns (regions #1 and #3) and 3 ns (regions #2 and #4) are visible.
Fig. 5
Fig. 5
Phase suppression applied to study an HT-1080 cell before the GM6001 inhibitor was washed out: (A) Phase suppression was applied to suppress the intensity from the intact biosensor in the image, leaving only intensity from the cleaved biosensor (mOrange2). (B) A set of detector phase angles was also selected to suppress the intensity from the cleaved biosensor (mOrange2). In this image, the result of that suppression strategy shows that the intact biosensor’s fluorescence is being emitted from a majority of the cell’s body. (C) A colour coding masked by the intensity image in (E) has been applied to the image. The green shading in the various pixels indicates where in the cell the fractional intensity of the cleaved biosensor (mOrange2) dominates the average intensity measured. (D) The pixels in which the fractional intensity is majority from the intact biosensor are coloured in red. This image has been masked by the intensity image in (E). (E) An intensity image of the cell is shown with three regions of interest indicated. (F) The pixels from each of the regions of interest in (E) are projected on the polar plot.
Fig. 6
Fig. 6
Analysis of an HT-1080 cell with phase suppression after the GM6001 inhibitor was washed out: (A) Two detector phases were chosen and images were acquired to suppress the intensity from the intact biosensor. As shown, intensity remained from the cleaved biosensor (mOrange2) along the edge of the cell. (B) Phase suppression was also used to suppress the intensity of the cleaved biosensor (mOrange2). When performed, the intensity from the intact biosensor was found predominantly in the interior of the cell. (C) Overlaid on the intensity image in (E) is a colour coding describing the locations where the majority of the average intensity measured in the pixel comes from the cleaved biosensor (mOrange2). (D) Coloured in red are the pixels in which the majority of the fractional intensity is from the intact biosensor. (E) An intensity image of the cell following the washout of GM6001 is shown along with three regions of interest indicated. (F). Three polar plots are shown in which the pixels from the regions of interest in (E) are projected.

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