Dynamic, Large-Scale Profiling of Transcription Factor Activity from Live Cells in 3D Culture

phenotypes. Taken together, our objective was to develop cellular arrays for dynamic, large-scale quantification of TF activity as cells organized into spherical structures within 3D culture.TF-specific and normalization reporter constructs were delivered in parallel to a cellular array containing a well-established breast cancer cell line cultured in Matrigel. Bioluminescence imaging provided a rapid, non-invasive, and sensitive method to quantify luciferase levels, and was applied repeatedly on each sample to monitor dynamic activity. Arrays measuring 28 TFs identified up to 19 active, with 13 factors changing significantly over time. Stimulation of cells with β-estradiol or activin A resulted in differential TF activity profiles evolving from initial stimulation of the ligand. Many TFs changed as expected based on previous reports, yet arrays were able to replicate these results in a single experiment. Additionally, arrays identified TFs that had not previously been linked with activin A.This system provides a method for large-scale, non-invasive, and dynamic quantification of signaling pathway activity as cells organize into structures. The arrays may find utility for investigating mechanisms regulating normal and abnormal tissue growth, biomaterial design, or as a platform for screening therapeutics

Stochastic light concentration from 3D to 2D reveals ultraweak chemi- and bioluminescence

For countless applications in science and technology, light must be concentrated, and concentration is classically achieved with reflective and refractive elements. However, there is so far no efficient way, with a 2D detector, to detect photons produced inside an extended volume with a broad or isotropic angular distribution. Here, with theory and experiment, we propose to stochastically transform and concentrate a volume into a smaller surface, using a high- albedo Ulbricht cavity and a small exit orifice through cavity walls. A 3D gas of photons produced inside the cavity is transformed with a 50% number efficiency into a 2D Lambertian emitting orifice with maximal radiance and a much smaller size. With high-albedo quartz-powder cavity walls ( P = 99.94%), the orifice area is 1/( 1 - P) approximate to 1600 times smaller than the walls' area. When coupled to a detectivity-optimized photon-counter ( D = 0.015 photon- 1 s1/ 2 cm) the detection limit is 110 photon s- 1 L- 1. Thanks to this unprecedented sensitivity, we could detect the luminescence produced by the non-catalytic disproportionation of hydrogen peroxide in pure water, which has not been observed so far. We could also detect the ultraweak bioluminescence produced by yeast cells at the onset of their growth. Our work opens new perspectives for studying ultraweak luminescence, and the concept of stochastic 3D/2D conjugation should help design novel light detection methods for large samples or diluted emitters

Development of a Lentivirus Vector-Based Assay for Non-Destructive Monitoring of Cell Fusion Activity

Cell-to-cell fusion can be quantified by endowing acceptor and donor cells with latent reporter genes/proteins and activators of these genes/proteins, respectively. One way to accomplish this goal is by using a bipartite lentivirus vector (LV)-based cell fusion assay system in which the cellular fusion partners are transduced with a flippase-activatable Photinus pyralis luciferase (PpLuc) expression unit (acceptor cells) or with a recombinant gene encoding FLPeNLS+, a nuclear-targeted and molecularly evolved version of flippase (donor cells). Fusion of both cell populations will lead to the FLPe-dependent generation of a functional PpLuc gene. PpLuc activity is typically measured in cell lysates, precluding consecutive analysis of one cell culture. Therefore, in this study the PpLuc-coding sequence was replaced by that of Gaussia princeps luciferase (GpLuc), a secretory protein allowing repeated analysis of the same cell culture. In myotubes the spread of FLPeNLS+ may be limited due to its nuclear localization signal (NLS) causing low signal outputs. To test this hypothesis, myoblasts were transduced with LVs encoding either FLPeNLS+ or an NLS-less version of FLPe (FLPeNLS-) and subsequently co-cultured in different ratios with myoblasts containing the FLPe-activatable GpLuc expression cassette. At different times after induction of cell-to-cell fusion the GpLuc activity in the culture medium was determined. FLPeNLS+ and FLPeNLS- both activated the latent GpLuc gene but when the percentage of FLPe-expressing myoblasts was limiting, FLPeNLS+ generally yielded slightly higher signals than FLPeNLS- while at low acceptor-to-donor cell ratios FLPeNLS- was usually superior. The ability of FLPeNLS+ to spread through myofibers and to induce reporter gene expression is thus not limited by its NLS. However, at high FLPe concentrations the presence of the NLS negatively affected reporter gene expression. In summary, a rapid and simple chemiluminescence assay for quantifying cell-to-cell fusion progression based on GpLuc has been developed