Evidence for annexin A6-dependent plasma membrane remodelling of lipid domains
Evidence for annexin A6-dependent plasma membrane remodelling of lipid domains
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DOI：
10.1111/bph.13022
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发表时间：
2015-04-01
影响因子：
7.3
通讯作者：
Rentero, Carles
中科院分区：
文献类型：
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作者：
Alvarez-Guaita, Anna;Vila de Muga, Sandra;Rentero, Carles
背景与目的膜联蛋白A6（Annexin A6，AnxA 6）是一种钙依赖性磷脂结合蛋白，可被募集到细胞膜上作为支架蛋白调节信号复合物的形成、胞内和胞外途径以及细胞胆固醇的分布。实验方法我们用Laurdan和di-4-ANEPPDHQ染色法分别在（i）人工膜、（ii）活细胞中观察膜的堆积和有序的脂相，并在（iii）细胞膜上观察膜的排列和有序的脂相。以及（iii）超分辨率成像（光活化定位显微术，PALM）和Ripley的K二阶点模式分析方法来评估AnxA 6如何调节质膜有序结构域和蛋白质聚集。纯化的AnxA 6诱导膜有序性的整体增加。然而，在活细胞中使用di-4-ANEPPDHQ的共聚焦显微镜显示，表达AnxA 6的细胞（其降低质膜胆固醇水平并修饰肌动蛋白细胞骨架网络）显示膜有序度降低（在A431和MEF细胞中分别约为15%和30%）。来自Lck 10和Src 15膜筏/非筏标记的PALM数据显示，AnxA 6表达诱导筏和非筏标记的聚类。Lck 10和Src 15在表达AnxA 6的细胞中的改变聚类也观察到胆固醇提取后与甲基-环糊精或肌动蛋白细胞骨架破坏与latrunculin B. ConclusionsAnxA 6诱导的质膜重塑表明，升高的AnxA 6表达降低膜秩序，通过调节细胞胆固醇稳态和肌动蛋白细胞骨架。这项研究提供了来自活细胞的第一个证据，支持目前膜联蛋白作为膜组织者的模型。
Background and PurposeAnnexin A6 (AnxA6) is a calcium-dependent phospholipid-binding protein that can be recruited to the plasma membrane to function as a scaffolding protein to regulate signal complex formation, endo- and exocytic pathways as well as distribution of cellular cholesterol. Here, we have investigated how AnxA6 influences the membrane order.Experimental ApproachWe used Laurdan and di-4-ANEPPDHQ staining in (i) artificial membranes; (ii) live cells to investigate membrane packing and ordered lipid phases; and (iii) a super-resolution imaging (photoactivated localization microscopy, PALM) and Ripley's K second-order point pattern analysis approach to assess how AnxA6 regulates plasma membrane order domains and protein clustering.Key ResultsIn artificial membranes, purified AnxA6 induced a global increase in membrane order. However, confocal microscopy using di-4-ANEPPDHQ in live cells showed that cells expressing AnxA6, which reduces plasma membrane cholesterol levels and modifies the actin cytoskeleton meshwork, displayed a decrease in membrane order (approximate to 15 and 30% in A431 and MEF cells respectively). PALM data from Lck10 and Src15 membrane raft/non-raft markers revealed that AnxA6 expression induced clustering of both raft and non-raft markers. Altered clustering of Lck10 and Src15 in cells expressing AnxA6 was also observed after cholesterol extraction with methyl--cyclodextrin or actin cytoskeleton disruption with latrunculin B.Conclusions and ImplicationsAnxA6-induced plasma membrane remodelling indicated that elevated AnxA6 expression decreased membrane order through the regulation of cellular cholesterol homeostasis and the actin cytoskeleton. This study provides the first evidence from live cells that support current models of annexins as membrane organizers.