High-Throughput Analysis of NF-κB Dynamics in Single Cells Reveals Basal Nuclear Localization of NF-κB and Spontaneous Activation of Oscillations

Abstract

NF-κB is a transcription factor that upon activation undergoes cycles of cytoplasmic-to-nuclear and nuclear-to-cytoplasmic transport, giving rise to so called “oscillations”. In turn, oscillations tune the transcriptional output. Since a detailed understanding of oscillations requires a systems biology approach, we developed a method to acquire and analyze large volumes of data on NF-κB dynamics in single cells. We measured the time evolution of the nuclear to total ratio of GFP-p65 in knock-in mouse embryonic fibroblasts using time-lapse imaging. We automatically produced a precise segmentation of nucleus and cytoplasm based on an accurate estimation of the signal and image background. Finally, we defined a set of quantifiers that describe the oscillatory dynamics, which are internally normalized and can be used to compare data recorded by different labs. Using our method, we analyzed NF-κB dynamics in over 2000 cells exposed to different concentrations of TNF- α α. We reproduced known features of the NF-κB system, such as the heterogeneity of the response in the cell population upon stimulation and we confirmed that a fraction of the responding cells does not oscillate. We also unveiled important features: the second and third oscillatory peaks were often comparable to the first one, a basal amount of nuclear NF-κB could be detected in unstimulated cells, and at any time a small fraction of unstimulated cells showed spontaneous random activation of the NF-κB system. Our work lays the ground for systematic, high-throughput, and unbiased analysis of the dynamics of transcription factors that can shuttle between the nucleus and other cell compartments.

Single-cell analyses reveal an attenuated NF-κB response in theSalmonella-infected fibroblast

“…26,27 Single-cell studies show that NF-kB presents a rich dynamics, including oscillations of the nuclear concentration. 25,[28][29][30] This type of dynamics has been observed for a number of transcription factors. 31 Most in vitro studies involving host-pathogen interactions have been performed using pooled cell cultures.…”

“…GFP-p65 MEF were incubated with DsRed-expressing S. Typhimurium for 10 min, a condition in which an average of »50% of fibroblasts harbor red intracellular bacteria. Nuclei of live cells were stained with a Hoechst derivative, which allowed automatic cell segmentation and tracking by our software, designed to quantify NF-kB nuclear translocation 30,37 (Fig. 1A, upper panel).…”

“…This NCI value has been used in a number of previous studies as direct measurement of NF-kB activation. 28,30,37,38 To distinguish infected (STC) and uninfected (ST-) cells in our time-lapse experiment, we calibrated an automatic detection method. For each cell, we determined the number of red pixels in the DsRed channel located in the cytoplasmic region.…”

Stochasticity and negative feedback lead to pulsed dynamics and distinct gene activity patterns

“…This motif regulates the activity of important transcription factors such as NF-κB [2] and p53 [3] and has been shown to give rise to pulses in the concentration of the proteins of the network (see e.g. [4,5]) as predicted by mathematical models [6]. The role of this pulsed dynamics is not fully understood though: theoretical studies suggest that they can give rise to a more reliable protein production [7] while experiments show that oscillatory dynamics can determine the cell fate [8].On the other hand, gene expression is an intrinsically noisy process [9] and models in which the gene state switches randomly between active and inactive states are able to fit well experimental data [10].…”

“…On the other hand, by using a simple peak detection algorithm we can detect the timing between two consecutive peaks T and their amplitude A peak . For these calculations we consider only peaks of at least 10% of A tot , the order of magnitude that can be detected in experiments of activator dynamics such as NF-κB [5]. The distributions of these magnitudes are shown in Fig.…”