Department of Chemistry, University of California, Riverside, Riverside, California 92521, USA.
Analyst. 2021 Sep 7;146(17):5307-5315. doi: 10.1039/d1an00751c. Epub 2021 Aug 5.
Analyzing intracellular signalling protein activities in living cells promises a better understanding of the signalling cascade and related biological processes. We have previously developed cyclic peptide-based probes for analyzing intracellular AKT signalling activities, but these peptide probes were not cell-permeable. Implementing fusogenic liposomes as delivery vehicles could circumvent the problem when analyzing adherent cells, but it remained challenging to study suspension cells using similar approaches. Here, we present a method for delivering these imaging probes into suspension cells using digitonin, which could transiently perforate the cell membrane. Using U87, THP-1, and Jurkat cells as model systems representing suspended adherent cells, myeloid cells, and lymphoid cells, we demonstrated that low concentrations of digitonin enabled a sufficient amount of probes to enter the cytosol without affecting cell viability. We further combined this delivery method with a microwell single-cell chip and interrogated the AKT signalling dynamics in THP-1 and Jurkat cells, followed by immunofluorescence-based quantitation of AKT expression levels. We resolved the cellular heterogeneity in AKT signalling activities and showed that the kinetic patterns of AKT signalling and the AKT expression levels were related in THP-1 cells, but decoupled in Jurkat cells. We expect that our approach can be adapted to study other suspension cells.
分析活细胞内的信号蛋白活性有望更好地理解信号级联和相关的生物过程。我们之前开发了基于环肽的探针来分析细胞内 AKT 信号活性，但这些肽探针不能穿透细胞。使用融合脂质体作为递送载体可以解决分析贴壁细胞时的问题，但使用类似的方法研究悬浮细胞仍然具有挑战性。在这里，我们提出了一种使用溶血磷脂酰胆碱将这些成像探针递送到悬浮细胞中的方法，它可以瞬时穿孔细胞膜。使用 U87、THP-1 和 Jurkat 细胞作为代表悬浮贴壁细胞、髓样细胞和淋巴样细胞的模型系统，我们证明了低浓度的溶血磷脂酰胆碱能够使足够数量的探针进入细胞质，而不会影响细胞活力。我们进一步将这种递送方法与微孔单细胞芯片相结合，检测了 THP-1 和 Jurkat 细胞中的 AKT 信号动力学，然后通过免疫荧光定量 AKT 表达水平。我们解析了 AKT 信号活性的细胞异质性，并表明 THP-1 细胞中 AKT 信号的动力学模式与 AKT 表达水平相关，但 Jurkat 细胞中则不相关。我们期望我们的方法可以适用于研究其他悬浮细胞。
