Nephrology Division, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts 02129.
Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114.
J Biol Chem. 2020 Apr 24;295(17):5761-5770. doi: 10.1074/jbc.RA119.012069. Epub 2020 Mar 9.
Over the last several years it has become clear that higher order assemblies on membranes, exemplified by signalosomes, are a paradigm for the regulation of many membrane signaling processes. We have recently combined two-color direct stochastic optical reconstruction microscopy (dSTORM) with the (Clus-DoC) algorithm that combines cluster detection and colocalization analysis to observe the organization of 5-lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) into higher order assemblies on the nuclear envelope of mast cells; these assemblies were linked to leukotriene (LT) C production. In this study we investigated whether higher order assemblies of 5-LO and FLAP included cytosolic phospholipase A (cPLA) and were linked to LTB production in murine neutrophils. Using two- and three-color dSTORM supported by fluorescence lifetime imaging microscopy we identified higher order assemblies containing 40 molecules (median) (IQR: 23, 87) of 5-LO, and 53 molecules (62, 156) of FLAP monomer. 98 (18, 154) molecules of cPLA were clustered with 5-LO, and 77 (33, 114) molecules of cPLA were associated with FLAP. These assemblies were tightly linked to LTB formation. The activation-dependent close associations of cPLA, FLAP, and 5-LO in higher order assemblies on the nuclear envelope support a model in which arachidonic acid is generated by cPLA in apposition to FLAP, facilitating its transfer to 5-LO to initiate LT synthesis.
在过去的几年中，很明显，膜上的高级组装体，例如信号体，是许多膜信号转导过程调节的范例。我们最近将双色直接随机光学重建显微镜（dSTORM）与（Clus-DoC）算法相结合，该算法结合了聚类检测和共定位分析，以观察 5-脂氧合酶（5-LO）和 5-脂氧合酶激活蛋白（FLAP）在肥大细胞核膜上组装成高级组装体的情况；这些组装体与白三烯（LT）C 的产生有关。在这项研究中，我们研究了 5-LO 和 FLAP 的高级组装体是否包含细胞质磷脂酶 A（cPLA），并与鼠中性粒细胞中 LTB 的产生有关。通过双和三色 dSTORM 支持荧光寿命成像显微镜，我们鉴定了包含 40 个分子（中位数）（IQR：23，87）的 5-LO 和 53 个分子（62，156）的 FLAP 单体的高级组装体。98（18，154）个 cPLA 分子与 5-LO 聚类，77（33，114）个 cPLA 分子与 FLAP 相关。这些组装体与 LTB 的形成紧密相关。cPLA、FLAP 和 5-LO 在核膜上的高级组装体中的激活依赖性紧密关联，支持这样一种模型，即 cPLA 在接近 FLAP 的位置产生花生四烯酸，促进其向 5-LO 的转移，以启动 LT 合成。
