Gibbons Elizabeth, Murri Michael, Grabner Amy, Moss Eric, Campbell Lauryl, Nelson Jennifer, Judd Allan M, Bell John D
Department of Physiology and Developmental Biology, Brigham Young University, Provo, UT 84602, USA.
Biochim Biophys Acta. 2014 Oct;1838(10):2607-14. doi: 10.1016/j.bbamem.2014.05.028. Epub 2014 Jul 3.
A diminution in the order of membrane lipids, which occurs during apoptosis, has been shown to correlate with increased membrane susceptibility to hydrolysis by secretory phospholipase A2. Studies with artificial membranes, however, have demonstrated that the relationship between membrane order and hydrolysis is more complex than suggested thus far by cell studies. To better resolve this relationship, this study focused on comparisons between increasing temperature and calcium ionophore as means of decreasing membrane order in S49 cells. Although these two treatments caused comparable changes in apparent membrane order as detected by steady-state fluorescence measurements, only ionophore treatment enhanced phospholipase activity. Experiments with exogenously-added phosphatidylserine indicated that the difference was not due to the presence of that anionic phospholipid in the outer membrane leaflet. Instead, analysis of the equilibration kinetics of various cationic membrane probes revealed that the difference could relate to the spacing of membrane lipids. Specifically, ionophore treatment increased that spacing while temperature only affected overall membrane order and fluidity. To consider the possibility that the distinction with ionophore might relate to the actin cytoskeleton, cells were stained with phalloidin and imaged via confocal microscopy. Ionophore caused disruption of actin fibers while increased temperature did not. This apparent connection between membrane hydrolysis and the cytoskeleton was further corroborated by examining the relationship among these events during apoptosis stimulated by thapsigargin.
细胞凋亡过程中发生的膜脂有序性降低，已被证明与分泌型磷脂酶A2对膜水解的敏感性增加相关。然而，对人工膜的研究表明，膜有序性与水解之间的关系比迄今为止细胞研究显示的更为复杂。为了更好地解析这种关系，本研究着重比较了升高温度和钙离子载体作为降低S49细胞膜有序性的手段。尽管通过稳态荧光测量检测到这两种处理导致表观膜有序性发生了相当的变化，但只有离子载体处理增强了磷脂酶活性。添加外源磷脂酰丝氨酸的实验表明，差异并非由于外膜小叶中存在该阴离子磷脂。相反，对各种阳离子膜探针平衡动力学的分析表明，差异可能与膜脂间距有关。具体而言，离子载体处理增加了间距，而温度仅影响整体膜有序性和流动性。为了考虑离子载体产生差异可能与肌动蛋白细胞骨架有关的可能性，用鬼笔环肽对细胞进行染色并通过共聚焦显微镜成像。离子载体导致肌动蛋白纤维破坏，而升高温度则没有。通过研究毒胡萝卜素刺激的细胞凋亡过程中这些事件之间的关系，进一步证实了膜水解与细胞骨架之间的这种明显联系。
