Program in Quantitative and Systems Biology, Department of Biomedical Engineering, Oregon Health & Science University, 2730 S Moody Ave., Portland, OR 97201, USA.
Cancer Early Detection Advanced Research Center, Knight Cancer Institute, Oregon Health & Science University, 2720 S Moody Ave., Portland, OR 97201, USA.
Biomolecules. 2022 Jul 26;12(8):1033. doi: 10.3390/biom12081033.
Recent work suggests that Ras small GTPases interact with the anionic lipid phosphatidylserine (PS) in an isoform-specific manner, with direct implications for their biological functions. Studies on PS-Ras associations in cells, however, have relied on immuno-EM imaging of membrane sheets. To study their spatial relationships in intact cells, we have combined the use of Lact-C2-GFP, a biosensor for PS, with multicolor super resolution imaging based on DNA-PAINT. At ~20 nm spatial resolution, the resulting super resolution images clearly show the nonuniform molecular distribution of PS on the cell membrane and its co-enrichment with caveolae, as well as with unidentified membrane structures. Two-color imaging followed by spatial analysis shows that KRas-G12D and HRas-G12V both co-enrich with PS in model U2OS cells, confirming previous observations, yet exhibit clear differences in their association patterns. Whereas HRas-G12V is almost always co-enriched with PS, KRas-G12D is strongly co-enriched with PS in about half of the cells, with the other half exhibiting a more moderate association. In addition, perturbations to the actin cytoskeleton differentially impact PS association with the two Ras isoforms. These results suggest that PS-Ras association is context-dependent and demonstrate the utility of multiplexed super resolution imaging in defining the complex interplay between Ras and the membrane.
最近的研究表明，Ras 小 GTPases 以同种型特异性的方式与阴离子脂质磷脂酰丝氨酸 (PS) 相互作用，这对它们的生物学功能有直接影响。然而，关于细胞中 PS-Ras 相互作用的研究依赖于膜片的免疫电子显微镜成像。为了在完整细胞中研究它们的空间关系，我们将 PS 的生物传感器 Lact-C2-GFP 与基于 DNA-PAINT 的多色超分辨率成像结合使用。在约 20nm 的空间分辨率下，得到的超分辨率图像清楚地显示了 PS 在细胞膜上的非均匀分子分布及其与 caveolae 的共富集，以及与未识别的膜结构的共富集。双色成像后进行空间分析表明，KRas-G12D 和 HRas-G12V 都在模型 U2OS 细胞中与 PS 共富集，这证实了先前的观察结果，但它们的关联模式存在明显差异。虽然 HRas-G12V 几乎总是与 PS 共富集，但 KRas-G12D 在大约一半的细胞中与 PS 强烈共富集，另一半细胞则表现出更温和的关联。此外，肌动蛋白细胞骨架的扰动对两种 Ras 同型与 PS 的关联有不同的影响。这些结果表明 PS-Ras 相互作用是上下文依赖的，并证明了多路复用超分辨率成像在定义 Ras 与膜之间复杂相互作用的实用性。
