Cell membrane stiffness is critical for cellular function, with cholesterol and sphingomyelin as pivot contributors. Current methods for measuring membrane stiffness are often invasive, ex situ, and slow in process, prompting the need for innovative techniques. Here, we present a fluorescence resonance energy transfer (FRET)-based protein sensor designed to address these challenges. The sensor consists of two fluorescent units targeting sphingomyelin and cholesterol, connected by a linker that responds to the proximity of these lipids. In rigid membranes, cholesterol and sphingomyelin are in close proximity, leading to an increased FRET signal. We utilized this sensor in combination with confocal microscopy to explore changes in plasma membrane stiffness under various conditions, including differences in osmotic pressure, the presence of reactive oxygen species (ROS) and variations in substrate stiffness. Furthermore, we explored the impact of SARS-CoV-2 on membrane stiffness and the distribution of ACE2 after attachment to the cell membrane. This tool offers substantial potential for future investigations in the field of mechanobiology.

中文翻译：

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使用基因编码 FRET 传感器对活细胞质膜硬度进行原位定量成像


细胞膜硬度对于细胞功能至关重要，其中胆固醇和鞘磷脂是关键贡献者。目前测量膜刚度的方法通常是侵入性的、非原位的且过程缓慢，因此需要创新技术。在这里，我们提出了一种基于荧光共振能量转移（FRET）的蛋白质传感器，旨在解决这些挑战。该传感器由两个针对鞘磷脂和胆固醇的荧光单元组成，通过对这些脂质的接近做出反应的连接体连接。在刚性膜中，胆固醇和鞘磷脂非常接近，导致 FRET 信号增强。我们利用该传感器与共焦显微镜相结合，探索各种条件下质膜刚度的变化，包括渗透压的差异、活性氧 (ROS) 的存在和基质刚度的变化。此外，我们还探讨了 SARS-CoV-2 对细胞膜硬度的影响以及 ACE2 附着在细胞膜上后的分布。该工具为机械生物学领域的未来研究提供了巨大的潜力。