Aho Brandon M, Wagner Dylan J, Gosse Julie A, Hess Samuel T
Department of Physics and Astronomy, University of Maine, Orono, Maine.
Department of Molecular and Biomedical Sciences, University of Maine, Orono, Maine.
Biophys J. 2025 Jul 5. doi: 10.1016/j.bpj.2025.07.004.
Mast cells mediate their immuno- and neuro-modulatory effects by releasing granules containing bioactive substances. Phosphatidylinositol 4,5-bisphosphate (PIP), enriched at the plasma membrane, is a key signaling lipid involved in numerous physiological functions including the calcium entry needed for antigen-stimulated mast cell degranulation. However, functional nanoscale PIP clustering and dynamics have not been previously investigated in immune cells. Using the pleckstrin homology domain from PLCδ (PH) tagged with photoswitchable fluorescent protein Dendra2, clustering was revealed in the mast cell model RBL-2H3, both fixed and live. We also discovered that live RBL-2H3 cells have PH clusters that evolve over timescales of ∼100 s. Additionally, the distribution of PIP, and specifically PIP clusters themselves, are disrupted upon addition of the cationic, lipidic drug cetylpyridinium chloride (CPC). CPC led to smaller, less dense, and more circular clusters. Furthermore, PH molecular mobility increased after the addition of CPC, suggesting interference of this drug with PH binding to PIP. In addition to this pharmacological relevance, the physiology of PIP clusters during functional stimulation by antigen was investigated. Antigen stimulation led to increased cluster size, which was counteracted by CPC. In live cells, PH density outside clusters was altered by CPC but not by antigen. CPC increased the proportion of regions of high-density PH compared to all other regions of the plasma membrane. Although PH diffusion was, interestingly, not affected by antigen, it was increased by CPC, particularly in lower-density regions. Under all live-cell dynamics observed, PH demonstrated confinement that was consistent with simulated diffusion within potential wells with an elliptical shape. These findings illuminate the nanoscale behavior of PIP in immune cells and the correlation of that behavior with cell function.
肥大细胞通过释放含有生物活性物质的颗粒来介导其免疫调节和神经调节作用。富集于质膜的磷脂酰肌醇4,5-二磷酸（PIP）是一种关键的信号脂质，参与多种生理功能，包括抗原刺激的肥大细胞脱颗粒所需的钙内流。然而，功能性纳米级PIP聚集和动力学此前尚未在免疫细胞中进行研究。使用标记有光开关荧光蛋白Dendra2的PLCδ的pleckstrin同源结构域（PH），在固定和活的肥大细胞模型RBL-2H3中均发现了聚集现象。我们还发现活的RBL-2H3细胞具有随约100秒时间尺度演变的PH簇。此外，添加阳离子脂质药物十六烷基吡啶氯化物（CPC）后，PIP的分布，特别是PIP簇本身会受到破坏。CPC导致簇更小、密度更低且更呈圆形。此外，添加CPC后PH分子迁移率增加，表明该药物干扰了PH与PIP的结合。除了这种药理学相关性外，还研究了抗原功能刺激期间PIP簇的生理学。抗原刺激导致簇尺寸增加，这被CPC抵消。在活细胞中，CPC改变了簇外PH的密度，但抗原未改变。与质膜的所有其他区域相比，CPC增加了高密度PH区域的比例。有趣的是，尽管PH扩散不受抗原影响，但它被CPC增加，特别是在低密度区域。在观察到的所有活细胞动力学情况下，PH表现出的限制与在椭圆形势阱内的模拟扩散一致。这些发现阐明了免疫细胞中PIP的纳米级行为以及该行为与细胞功能的相关性。
