Abstract
Cell communication coordinates developmental processes, maintains homeostasis, and contributes to disease. Therefore, understanding the relationship between cells in a shared environment is crucial. Here we introduce Positive Ultra-bright Fluorescent Fusion For Identifying Neighbours (PUFFFIN), a cell neighbour-labelling system based upon secretion and uptake of positively supercharged fluorescent protein s36GFP. We fused s36GFP to mNeonGreen or to a HaloTag, facilitating ultra-bright, sensitive, colour-of-choice labelling. Secretor cells transfer PUFFFIN to neighbours while retaining nuclear mCherry, making identification, isolation, and investigation of live neighbours straightforward. PUFFFIN can be delivered to cells, tissues, or embryos on a customisable single-plasmid construct composed of interchangeable components with the option to incorporate any transgene. This versatility enables the manipulation of cell properties, while simultaneously labelling surrounding cells, in cell culture or in vivo. We use PUFFFIN to ask whether pluripotent cells adjust the pace of differentiation to synchronise with their neighbours during exit from naïve pluripotency. PUFFFIN offers a simple, sensitive, customisable approach to profile non-cell-autonomous responses to natural or induced changes in cell identity or behaviour.
Keywords:
HaloTag; Neighbour Labelling; PUFFFIN; Pluripotency; Synthetic Signalling.