Visualizing cellular phosphoinositide pools with GFP-fused protein-modules.
Visualizing cellular phosphoinositide pools with GFP-fused protein-modules.
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DOI：
10.1126/stke.2002.125.pl3
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发表时间：
2002-03-26
期刊：
影响因子：
--
通讯作者：
Varnai, Peter
中科院分区：
文献类型：
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作者：
Balla, Tamas;Varnai, Peter
肌醇磷脂因其作为响应各种刺激而产生的重要第二信使的前体在钙信号传导中所起的关键作用而广为人知。然而，在过去10年中，肌醇磷脂也已成为几乎存在于真核细胞的每一层膜中的通用信号成分。这些脂质由众多的肌醇磷脂激酶和磷酸酶在局部产生和降解，以控制特定膜区室中蛋白质信号复合物的募集和活性。细胞内肌醇磷脂变化所受到的时空限制给寻找能够检测此类变化的实验技术带来了新的挑战。利用进化选择的能够识别细胞磷酸肌醇的蛋白质结构域，我们通过将这些结构域与改进版的绿色荧光蛋白（EGFP）融合，创造了荧光分子；这些融合蛋白在活细胞内的分布可以被追踪，从而反映磷酸肌醇的变化。尽管该技术是少数能够以亚细胞分辨率提供活细胞中磷酸肌醇动态信息的技术之一，并且迅速得到广泛应用，但在解释数据时也需要考虑到它的局限性。在此，我们总结了我们在设计和使用这些构建体方面的经验，并阐述了我们对于解释通过该技术所获得数据的观点。
Inositol phospholipids are well known for their pivotal role in calcium signaling as precursors of important second messengers generated in response to various stimuli. However, over the last 10 years, inositides have also emerged as universal signaling components present in virtually every membrane of eukaryotic cells. These lipids are locally produced and degraded by the numerous inositide kinase and phosphatase enzymes, to control the recruitment and activity of protein signaling complexes in specific membrane compartments. The spatial and temporal constraints imposed on changes in cellular inositides pose new challenges in finding experimental techniques through which such changes can be examined. Taking advantage of the protein domains selected by evolution to recognize cellular phosphoinositides, we have created fluorescent molecules by fusing these domains to the improved version of green fluorescent protein (EGFP); the distribution of these fusion proteins can be followed within live cells, thereby reporting on changes in phosphoinositides. Although this technique is one of the few that provide information on phosphoinositide dynamics in live cells with subcellular resolution and has rapidly gained popularity, it also has limitations that need to be taken into account when interpreting the data. Here, we summarize our experience in designing and using these constructs and review our position concerning the interpretation of the data obtained by this technique.