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.

中文翻译：

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通过冻结断裂复制标记定义磷脂酰肌醇 4,5-二磷酸分布。


磷脂酰肌醇 4,5-二磷酸 [PtdIns（4,5）P2] 是质膜功能所必需的磷脂，但其二维分布尚不清楚。在这里，我们将十二烷基硫酸钠处理的快速冷冻细胞的冷冻断裂复制标记 （SDS-FRL） 的结果与实际的 PtdIns（4,5）P2 含量以及通过荧光生物传感器和化学固定膜标记获得的结果进行了比较。在酵母中，SDS-FRL 明显表明 PtdIns（4,5）P2 在 Can1 （MCC）/内异体的膜区室中富集，尤其是在弯曲的 MCC/内异体中，但荧光生物传感器 GFP-PLC1δ-PH 则不明显。荧光生物传感器无法检测到急性 ATP 耗竭后剩余的 PtdIns（4,5）P2 和静止期分别为对照水平的 30.0% 和 56.6%，而 SDS-FRL 的标记强度反映了 PtdIns（4,5）P2 量。在 PC12 细胞中，PtdIns（4,5）P2 在甲醛固定质膜中以点状模式观察到，而它通过 SDS-FRL 随机分布，并在甲醛固定后显示聚集。结果表明，速冻细胞的 SDS-FRL 可以最可靠地定义 PtdIns（4,5）P2 的分布。