A High-Content Assay for Biosensor Validation and for Examining Stimuli that Affect Biosensor Activity

Abstract

Biosensors are valuable tools used to monitor many different protein behaviors in vivo. Demand for new biosensors is high, but their development and characterization can be difficult. During biosensor design, it is necessary to evaluate the effects of different biosensor structures on specificity, brightness, and fluorescence responses. By co-expressing the biosensor with upstream proteins that either stimulate or inhibit the activity reported by the biosensor, one can determine the difference between the biosensor's maximally activated and inactivated state, and examine response to specific proteins. We describe here a method for biosensor validation in a 96-well plate format using an automated microscope. This protocol produces dose-response curves, enables efficient examination of many parameters, and unlike cell suspension assays, allows visual inspection (e.g., for cell health and biosensor or regulator localization). Optimization of single-chain and dual-chain Rho GTPase biosensors is addressed, but the assay is applicable to any biosensor that can be expressed or otherwise loaded in adherent cells. The assay can also be used for purposes other than biosensor validation, using a well-characterized biosensor as a readout for effects of upstream molecules.

Keywords: FRET; HC screening; biosensor; fluorescence; microplate assay.

Figure 1

The Rac1 FLARE.dc biosensor. When Rac1 is in the GDP-bound state, it has low affinity for the p21 binding domain of PAK1 (PBD). The two peptides are unassociated and there is negligible FRET from the CyPet on Rac1 to the YPet on the PBD. When in the GTP-bound state, Rac1 has increased affinity for the PBD and interacts with it, bringing the CyPet and YPet tags into proximity and allowing FRET to take place. Rac1 can be converted from the GDP-bound to the GTP-bound state by GEFs, and back to the GDP-bound state by GAPs. The GDP-bound GTPase can also be complexed with GDI, which sequesters Rac1 in the cytoplasm and prohibits interaction with the PBD.

Figure 2

Regulator expression increases nearly linearly with the mass of DNA transfected over a wide range of DNA mass. Samples transfected with varying dosage of expression plasmid encoding mCherry-tagged regulator (filled circles: RhoGDI1; empty circles: active Vav2 GEF) were imaged and total mCherry emission, after background subtraction, was plotted against transfected regulator plasmid mass.

Figure 3

Layout of cell transfections in black-walled, flat-bottom imaging 96-well microplate. Column 1 contains 8 replicates of mock transfected cells. Column 2 contains four replicate wells each of cells transfected with a donor-only or acceptor-only control. The remainder of the columns contain cells transfected with biosensor plus varying amounts of regulator. Regulator amount halves serially from right to left across row A, and then again across row B, to cover a dosage range of greater than 200,000 fold. Subsequent rows contain replicates of rows A and B, for a total of four replicate wells for each biosensor/regulator combination. M = mock. D = donor only. A = Acceptor only. X = 4.26. B = biosensor. Y = 42.56. R = regulator.

Figure 4

Layout of Lipofectamine/DNA transfection reactions in v-bottom 96-well microplates. M = mock. D = donor only. A = Acceptor only. X = 20. B = biosensor. Y = 200. R = regulator.

Figure 5

DNA solutions prepared for transfection reactions. Volumes that are added to the solutions are highlighted in gray.

Figure 6

Distribution of Figure 5 DNA solutions into v-bottom microplate prior to serial dilution.

Figure 7

Ratiometric FRET indices. A = acceptor bleedthrough correction, acceptor bleedthrough coefficient, acceptor-only sample, or acceptor channel, depending on the column heading. D = donor bleedthrough correction, donor bleedthrough coefficient, donor-only sample, or donor channel, depending on the column heading. F = FRET channel. Inter = intermolecular biosensor design. Intra = intramolecular biosensor design. An equal sign means “equivalent”. M = monotonic. L = linear. P = proportional. IL = inversely linear. NA = not applicable.

Figure 8

Biosensor response to regulator expression. The Rac1 FLARE.dc biosensor or a donor-only control biosensor were transfected into LinXE cells along with varying amounts of active Vav2 GEF or RhoGDI1 regulators. For each well, the sensitized emission:donor emission ratio was calculated and normalized by dividing by the donor bleedthrough coefficient. Plotted are mean ratio values for two replicate wells of each transfection condition with error bars representing standard deviations, as a function of regulator plasmid mass transfected.

Figure 9

Checking for consistency in the background measurements. Mock transfected wells, which are used to determine background values for each channel, were imaged, and the sum of pixel intensities were computed in all channels. The resulting values for the Donor channel are shown plotted against the well replicate number. One well appears to have an outlying value, but this will not affect the median value, which will be calculated and used for background subtraction of other wells.

Figure 10

Checking for consistency in bleedthrough coefficient measurements. Donor-only transfected wells, which are used to calculate donor bleedthrough coefficients, were imaged, and the FRET:donor ratio (equivalent to the donor bleedthrough coefficient in the case of donor-only control wells) was plotted versus replicate number. There is very little variance across the replicates.

Figure 11

A Rac1 biosensor was transfected into LinXE cells with a gradient of Vav2 GEF, using different transfection conditions as listed in the legend. The amount of Lipofectamine, the amount of Plus Reagent, and the order of addition (Lipofectamine added either before or after DNA titration) were varied. Mean values of a FRET index (error bars are standard deviations of four replicate wells) were plotted against transfected Vav2 plasmid mass.

Figure 12

A typical image of Rac1 FLARE.dc biosensor expressed in LinXE cells (contrast enhanced).