PUFFFIN: A novel, ultra-bright, customisable, single-plasmid system for labelling cell neighbourhoods
PUFFFIN: A novel, ultra-bright, customisable, single-plasmid system for labelling cell neighbourhoods
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DOI：
10.1101/2023.09.06.556381
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发表时间：
2023-09
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影响因子：
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通讯作者：
Tamina Lebek;Mattias Malaguti;Alistair Elfick;S. Lowell
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作者：
Tamina Lebek;Mattias Malaguti;Alistair Elfick;S. Lowell
细胞通讯协调发育、体内平衡和疾病。理解这些过程取决于有效的方法来研究细胞如何影响它们的邻居。在这里，我们提出了正超亮荧光融合识别邻居（PUFFFIN），这是一种基于分泌和摄取正增压荧光蛋白s36GFP的新型细胞邻居标记系统。为了实现敏感标记，我们通过将s36GFP融合到超亮蛋白mNeonGreen中来放大荧光信号。分泌细胞将s36GFP-mNeonGreen转移到邻近细胞，同时保留一个核mCherry，使其直接明确地识别、分离和分析分泌细胞的邻近细胞。s36GFP-mNeonGreen融合的转移在几分钟内发生，即使是相对短暂的相互作用也可以通过实时成像或流式细胞术捕获或跟踪。为了提高使用的灵活性，我们采用了HaloTag技术，以方便选择颜色标签。为了进一步增加应用范围，我们设计了一种可定制的单质粒结构，由可互换的组件组成，可选择加入任何额外的转基因。这允许用户操作细胞的属性，同时将荧光标签应用于周围的细胞。PUFFFIN提供了一种简单、敏感、可定制的方法来分析哺乳动物细胞对细胞身份或行为的自然或诱导变化的非细胞自主反应。图形抽象
Cell communication orchestrates development, homeostasis, and disease. Understanding these processes depends on effective methods to study how cells influence their neighbours. Here, we present Positive Ultra-bright Fluorescent Fusion For Identifying Neighbours (PUFFFIN), a novel cell neighbour-labelling system based upon secretion and uptake of a positively supercharged fluorescent protein s36GFP. To achieve sensitive labelling, we amplified the fluorescent signal by fusing s36GFP to the ultra-bright protein mNeonGreen. Secretor cells transfer s36GFP-mNeonGreen to neighbours while retaining a nuclear mCherry, making it straightforward to unambiguously identify, isolate, and profile neighbours of secretor cells. Transfer of the s36GFP-mNeonGreen fusion occurs within minutes, enabling even relatively transient interactions to be captured or tracked over time with live-imaging or flow cytometry. To enhance flexibility of use, we incorporated HaloTag technology to facilitate colour- of-choice labelling. To further increase the range of applications, we engineered a customisable single-plasmid construct composed of interchangeable components with option to incorporate any additional transgene. This allows users to manipulate the properties of a cell, while at the same time applying a fluorescent label to the surrounding cells. PUFFFIN offers a simple, sensitive, customisable approach to profile non-cell-autonomous responses to natural or induced changes in cell identity or behaviour in mammalian cells. Graphical abstract