TNFR1 mediates heterogeneity in single-cell NF-κB activation.

Nuclear factor kappa B (NF-κB) is a key regulator in immune signaling and is known to exhibit a digital activation pattern. Yet the molecular basis underlying the heterogeneity in NF-κB activation at single-cell level is not entirely understood. Here, we show that NF-κB activation in single cells is largely regulated by intrinsic differences at the receptor level. Using the genome editing and time-lapse imaging, we directly characterize endogenous TNFR1 dynamics and NF-κB activation from the same single cells. Total internal reflection fluorescence (TIRF) microscopy shows that endogenous TNFR1 forms pre-ligand clusters in the resting cells. Upon tumor necrosis factor (TNF) stimulation, the diffusion coefficient of membrane TNFR1 was significantly decreased and a substantial level of TNFR1 undergoes oligomerization to form trimers and hexamers. Moreover, multi-color cell imaging reveals that both digital and graded information processing regulate NF-κB activation across different TNFR1 expression levels. Our results indicate that single-cell NF-κB activation potential strongly correlates with its TNFR1 characteristics.

Chip assisted formation of phase-separated liposomes for reconstituting spatial protein-lipid interactions.

Spatially organized molecular interactions are fundamental features underlying many biochemical processes in cells. These spatially defined reactions are essential to ensure high signaling specificity and are indispensable for maintaining cell functions. The construction of synthetic cell models that can resemble such properties is thus important yet less investigated. In this study, we present a reliable method for the rapid production of highly uniform phase-separated liposomes as synthetic cell models. Specifically, a microfluidics-based strategy coupled with custom reagents for generating size-tunable liposomes with various lipid compositions is presented. In addition, an important cell signaling interacting pair, the pleckstrin homology (PH) domain and PIP2 lipid, is used to demonstrate the controlled molecular assembly inside these liposomes. The result shows that PIP2 on phase-separated domains successfully recruits the PH domains to realize spatially defined molecular interactions. Such a system is versatile and can be expanded to synthesize other proteins for realizing multiplexed molecular interactions in the same liposome. Phase-separated lipid domains can also be used to recruit targeted proteins to initiate localized reactions, thus paving the way for organizing a complex signaling cascade in the synthetic cell.