Fluorescent tools to analyse peroxisome-ER interactions in mammalian cells.
Fluorescent tools to analyse peroxisome-ER interactions in mammalian cells.
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DOI：
10.1177/2515256419848641
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发表时间：
2019-06-05
期刊：
影响因子：
--
通讯作者：
Schrader, Michael
中科院分区：
文献类型：
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作者：
Bishop, Alexa;Kamoshita, Maki;Schrader, Michael
过氧化物酶体和内质网（ER）在脂质相关的代谢途径中广泛合作，ER还提供磷脂，使过氧化物酶体膜在分裂前扩张。最近，我们确定了过氧化物酶体蛋白ACBD 5和ACBD 4，以及ER蛋白VAPB作为束缚组分，其物理相互作用以促进膜接触位点处的过氧化物酶体-ER缔合。这些系链蛋白的过表达或丢失改变了过氧化物酶体-ER相互作用的程度，影响了这两个隔室之间的脂质交换。为了促进进一步研究过氧化物酶体-ER协会在膜接触位点的水平，其作用，组成和调节，我们已经开发了两个基于荧光的系统来监测过氧化物酶体-ER相互作用。我们修改了邻位连接试验和分裂荧光报告系统使用分裂superfolder绿色荧光蛋白。使用邻近连接试验，我们能够测量过氧化物酶体-ER相互作用的变化，而分裂荧光报告基因则更加有限，仅允许我们标记ER-过氧化物酶体接触。我们表明，这两种技术可以是有用的工具包的方法来研究过氧化物酶体ER协会和探索各自的相对优点。
Peroxisomes and the endoplasmic reticulum (ER) cooperate extensively in lipid-related metabolic pathways, and the ER also provides phospholipids to enable the peroxisomal membrane to expand prior to division. Recently, we identified peroxisomal proteins ACBD5 and ACBD4, and the ER protein VAPB as tethering components which physically interact to foster peroxisome-ER associations at membrane contact sites. Overexpression or loss of these tether proteins alters the extent of peroxisome-ER interactions, impacting on lipid exchange between these two compartments. To facilitate further studies into peroxisome-ER associations at the level of membrane contact sites, their role, composition and regulation, we have developed two fluorescence-based systems to monitor peroxisome-ER interactions. We modified a proximity ligation assay and a split-fluorescence reporter system using split superfolder green fluorescent protein. Using the proximity ligation assay we were able to measure changes in peroxisome-ER interactions whilst the split-fluorescence reporter was more limited and only allowed us to label ER-peroxisome contacts. We show that both techniques can be useful additions to the toolkit of methods to study peroxisome-ER associations and explore the relative merits of each.