Key Laboratory of the Ministry for Advanced Catalysis Materials, College of Chemistry and Materials Science, Zhejiang Normal University, Jinhua, 321004, PR China.
College of Life Sciences, Zhejiang Normal University, Jinhua, 321004, PR China.
Talanta. 2024 Nov 1;279:126583. doi: 10.1016/j.talanta.2024.126583. Epub 2024 Jul 17.
The plasma membrane involves in many important biological events such as cell fusion and programmed cell death, but most of current plasma membrane probes cannot meet the requirement of long-term specific anchoring to the plasma membrane. Herein, we propose a molecular side-chain engineering strategy to modulate the long-term imaging performance of fluorescent dyes to the plasma membrane by regulating the cell permeability and anchoring ability. A series of FMR dyes with different lengths of side chains were designed and synthesized, and their transmembrane behaviours and staining performance were evaluated in living HeLa cells. We found that short-chain and medium-chain FMR dyes have excellent cell permeability without the labeling ability to the plasma membrane while the long-chain FMR dyes specifically stain the plasma membrane and can be firmly anchored to the plasma membrane for a long period of time. These long-chain FMR dyes have high stain specificality to the plasma membrane, and C10-FMR can be anchored to the plasma membrane of living cells for 2 h, which enables it to continuously monitor dynamic changes of the plasma membrane. The three-dimensional precision imaging of various cells was achieved using C10-FMR, which provides an opportunity to obtain complete information on the three-dimensional spatial morphology of the plasma membrane. The PEG-induced cell fusion of chicken red blood cells and HO-induced apoptosis of HeLa cells were monitored by real-time tracking of dynamic changes of the plasma membrane during these processes, which provide solid examples to prove the usefulness of these fluorescent dyes as long-term imaging tools. This work validates the hypothesis that cell permeability of membrane dyes can be readily regulated by tuning the side chains, and provides the effective design strategy of fluorescent dyes for 3D and long-term dynamic tracking of the plasma membrane of diverse animal cells.
质膜参与许多重要的生物学事件，如细胞融合和程序性细胞死亡，但大多数现有的质膜探针不能满足长期特异性锚定质膜的要求。在此，我们提出了一种分子侧链工程策略，通过调节细胞膜的通透性和锚定能力来调节荧光染料对质膜的长期成像性能。设计并合成了一系列具有不同侧链长度的 FMR 染料，并在活 HeLa 细胞中评价了它们的跨膜行为和染色性能。我们发现短链和中链 FMR 染料具有优异的细胞通透性，而没有对质膜的标记能力，而长链 FMR 染料特异性地染色质膜，并能长时间牢固地锚定在质膜上。这些长链 FMR 染料对质膜具有高的染色特异性，C10-FMR 可以锚定在活细胞的质膜上 2 小时，这使其能够持续监测质膜的动态变化。使用 C10-FMR 实现了各种细胞的三维精确定位成像，为获得质膜三维空间形态的完整信息提供了机会。通过实时跟踪这些过程中质膜动态变化，监测了鸡红细胞的 PEG 诱导融合和 HeLa 细胞的 HO 诱导凋亡，为这些荧光染料作为长期成像工具的有用性提供了确凿的实例。这项工作验证了通过调整侧链可以轻松调节膜染料细胞通透性的假设，并为不同动物细胞的质膜的 3D 和长期动态追踪提供了有效的荧光染料设计策略。
