Balla Tamas, Várnai Péter
Endocrinology and Reproduction Research Branch, National Institute of Child Health & Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Sci STKE. 2002 Mar 26;2002(125):pl3. doi: 10.1126/stke.2002.125.pl3.
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.
肌醇磷脂作为重要第二信使的前体，在钙信号传导中发挥关键作用，这一点广为人知，这些第二信使是细胞对各种刺激做出反应时产生的。然而，在过去十年中，肌醇磷脂也已成为真核细胞几乎每个膜中都存在的通用信号成分。这些脂质在局部由众多肌醇磷脂激酶和磷酸酶产生和降解，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挑战。利用进化过程中选择的用于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绿色荧光蛋白（EGFP）的改进版本融合，创建了荧光分子；这些融合蛋白的分布可在活细胞内追踪，从而反映磷酸肌醇的变化。尽管这项技术是少数能够在亚细胞分辨率下提供活细胞中磷酸肌醇动态信息的技术之一，并且迅速受到欢迎，但在解释数据时，它也有一些需要考虑的局限性。在此，我们总结了设计和使用这些构建体的经验，并回顾了我们对通过该技术获得的数据的解释立场。
