2.7. BlaM Assay Spectral and Fluorescence Lifetime Image Analysis

To rule out artifacts due to photo-bleaching and insufficient signal to noise, only cells with at least 100–1000 photons per pixel and negligible amount of bleaching were included in the analysis after a 3 × 3 image binning. The acquired fluorescence decay of each pixel was deconvoluted with the instrument response function (IRF) and fitted with two-exponential theoretical models using Leica Application Suite X (LAS X) from Leica Microsystems. The fraction of interacting donor (f_D) was calculated using Leica Application Suite X (LAS X) from Leica Microsystems and ImageJ software (https://imagej.nih.gov/ij/) following Equations (2) and (3) (below). The ratio of average intenstity was then calculated pixel by pixel using ImageJ software. Regions of interest corresponding to individual cells were selected using a semi-automated macro using Image J software and applied to calculate the average intensity ratio and fraction of interacting donor per cell. Individual values per cell were plotted after normalization to the threshold given by our negative control (No Env virions packaging Vpr-BlaM).

One can define the catalytic reaction of the Vpr-BlaM cleaving the CCF2-AM substrate right after HIV-1 fusion as:

where E is the enzyme, in this case the Vpr-BlaM, S the substrate, in this case the CCF2-AM, and P the products (cleaved CCF2-AM, hydroxycoumarin + fluorescein, Figure 1). The Michaelis–Menten constant, K_M, is defined as:

Schema of the β-lactamase (BlaM) assay applied to study human immunodeficiency virus type 1 (HIV-1) fusion utilizing intensity-based Förster resonance energy transfer (FRET) approaches and FRET fluorescence lifetime imaging microscopy (FLIM). HIV-1 pseudoparticles bearing the Vpr-BlaM are exposed to live cells expressing CD4 and co-receptors so that the HIV-1 particles are allowed to fuse with the host membrane and release their capsid into the host cytosol. The BlaM enzyme reaches the CCF2-AM FRET biosensor composed of hydroxycoumarin linked to fluorescein by the BlaM recognition domain. Once the BlaM enzyme starts the catalytic reaction, CCF2-AM is cleaved into hydroxycoumarin (donor) and fluorescein (acceptor) and FRET is disrupted. When employing FLIM, one can apply a two-exponential method that takes into account the situation in which not all fluorophore is engaged in FRET. A change from green to blue is also observed when looking at the visible emission spectra of CCF2-AM during the BlaM enzymatic reaction. The green emission (~520 nm) when exciting hydroxycoumarin at 405 nm comes from FRET; when CCF2-AM is cleaved hydroxycoumarin emits in the blue (~447 nm).

It has been shown [11] that there is a linear dependence between K_M and [E] which linearly scales with [E] with a –1 slope in live cells. This experimental relationship led us to propose a linear model for this catalytic reaction that we could recapitulate in Figure 2. Importantly, Zotter and coleagues [11] showed that the slower diffusion coeffcient of the substrate (CCF2-AM) in the cytosol of live cells as compared to in-solution is the explanantion for this linear behavior. Both hydroxycourmarin and fluorescein should have similar diffusion coeficients regardless of the cell line utilized. In [11] it was also shown that the metabolic state of HeLa cells did not perturb this linear dependency nor the BlaM efficiency.

BlaM HIV-1 fusion calibration utilizing intensity-based FRET approaches and FRET-FLIM. (A) Images of the BlaM assay in TZM-bl cells exposed to different MOIs (1, 2, 5 and 10) of HIV-1_JFRL virions, analyzed using FRET intensity ratio and the fraction of interacting donor (f_D). Images are pseudocolored in red (fusion negative) and green (fusion positive). Scale bars are 20 µm. The statistics coming from the two methods, (B) FRET intensity and (C) f_D, are presented in which every dot represents a single cell (n > 50 cells per condition from one experiment). The green dotted line represents the threshold taken from TZM-bl cells exposed to HIV-1 without spikes (No Env) virions. (D,E) The calibration curves for both methods are presented together with the linear regression and equation/s. The calibration curves were obtained from the data points of three independent experiment (Exp. 1–3). Each symbol represents the mean and SD per condition, per experiment.

In FLIM, for a single fluorophore in a homogeneous environment the fluorescecnce decay can be defined in the following way:

where the [S^*] corresponds to hydroxycoumarin (the donor in the CCF2-AM complex, with fluorescein being the acceptor) in the excited state (right after excitation with the pulsed 440 nm laser there is a promotion of one photon from the hydroxycoumarin higher occupied molecular orbital (HOMO) to the lowest unoccupied hydroxycoumarin molecular orbital (LUMO)). k_r is the kinetic constant of photon relaxation, which, in turn, emits light between the LUMO and the HOMO [12]. Finally, τ is the lifetime (in ns) for the photons residing in the LUMO.

According to the BlaM catalytic equation (Equation (1)), one can define a proportion of substrate (CCF2-AM) engaged in FRET (f_D) and a proportion that is not (1 – f_D) and define the next equation:

For Equations (3) and (4), τ is the lifetime, τ_D the CCF2-AM donor lifetime, τ_F the FRET lifetime and f_D the fraction of CCF2-AM donor engaged in FRET right before cleavage (Figure 1). Time domain FLIM allows the simultaneous calculation of the fluorescence lifetime of the donor, the FRET lifetime, which implies E and f_D; which in this case is the key parameter as f_D is proportional to [ES]₀ or uncleaved CCF2-AM. We therefore employed Equation (4) as a model to analyze all FLIM CCF2-AM images and recover f_D pixel by pixel. The averaged f_D per cell was then recovered and plotted against the MOI to recover the linear dependency for the CCF2-AM BlaM catalytic reaction.