Research Institute for Diseases of Old Age, Juntendo University Graduate School of Medicine , Tokyo, Japan.
Medical Research Institute, Tokyo Medical and Dental University , Tokyo, Japan.
J Cell Biol. 2025 Jan 6;224(1). doi: 10.1083/jcb.202311067. Epub 2024 Nov 4.
Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is a phospholipid essential for plasma membrane functions, but its two-dimensional distribution is not clear. Here, we compared the result of sodium dodecyl sulfate-treated freeze-fracture replica labeling (SDS-FRL) of quick-frozen cells with the actual PtdIns(4,5)P2 content and the results obtained by fluorescence biosensor and by labeling of chemically-fixed membranes. In yeast, enrichment of PtdIns(4,5)P2 in the membrane compartment of Can1 (MCC)/eisosome, especially in the curved MCC/eisosome, was evident by SDS-FRL, but not by fluorescence biosensor, GFP-PLC1δ-PH. PtdIns(4,5)P2 remaining after acute ATP depletion and in the stationary phase, 30.0% and 56.6% of the control level, respectively, was not detectable by fluorescence biosensor, whereas the label intensity by SDS-FRL reflected the PtdIns(4,5)P2 amount. In PC12 cells, PtdIns(4,5)P2 was observed in a punctate pattern in the formaldehyde-fixed plasma membrane, whereas it was distributed randomly by SDS-FRL and showed clustering after formaldehyde fixation. The results indicate that the distribution of PtdIns(4,5)P2 can be defined most reliably by SDS-FRL of quick-frozen cells.
磷脂酰肌醇 4,5-二磷酸[PtdIns(4,5)P2]是一种对质膜功能至关重要的磷脂，但它的二维分布尚不清楚。在这里，我们比较了快速冷冻细胞的十二烷基硫酸钠处理冷冻断裂复型标记（SDS-FRL）的结果与实际 PtdIns(4,5)P2 含量以及荧光生物传感器和化学固定膜标记的结果。在酵母中，Can1（MCC）/内体的膜隔室中 PtdIns(4,5)P2 的富集，特别是在弯曲的 MCC/内体中，通过 SDS-FRL 明显，但通过荧光生物传感器 GFP-PLC1δ-PH 则不明显。急性 ATP 耗竭后和静止期分别剩余 30.0%和 56.6%的对照水平的 PtdIns(4,5)P2 不能通过荧光生物传感器检测到，而 SDS-FRL 的标记强度反映了 PtdIns(4,5)P2 的数量。在 PC12 细胞中，甲醛固定的质膜中 PtdIns(4,5)P2 呈点状模式，而 SDS-FRL 显示随机分布，并在甲醛固定后出现聚类。结果表明，通过快速冷冻细胞的 SDS-FRL 可以最可靠地定义 PtdIns(4,5)P2 的分布。
