PUFFFIN: an ultra-bright, customisable, single-plasmid system for labelling cell neighbourhoods.

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. Synopsis: While there are some options for labeling cellular neighbours, they often have low flexibility, require modification of multiple cells with different plasmids, or are otherwise technically challenging. To overcome these limitations, this study develops Positive Ultra-bright Fluorescent Fusion For Identifying Neighbours (PUFFFIN), a method based on secretion and receptor-independent uptake of a positively charged fluorescent fusion molecule. PUFFFIN uses a universal uptake mechanism to deliver fluorescent label to neighbouring cells without the need to engineer them with label-uptake machinery. All PUFFFIN components are encoded on a single modular plasmid, amenable to delivery by transfection, electroporation, or lentivirus. PUFFFIN enables ultra-bright colour-of-choice labelling for unambiguous and rapid labelling of neighbours in cell culture or in vivo. PUFFFIN labelling does not compromise viability, enabling functional analysis of live neighbours. PUFFFIN labelling is used to demonstrate that pluripotent cells adjust the pace of differentiation to synchronise with their neighbours. Positive Ultra-bright Fluorescent Fusion For Identifying Neighbours, or PUFFFIN, allows visualization and isolation of surrounding cells in culture, tissues, or embryos to profile non-cell-autonomous responses. [ABSTRACT FROM AUTHOR]