期刊
BMC CELL BIOLOGY
卷 10, 期 -, 页码 -出版社
BIOMED CENTRAL LTD
DOI: 10.1186/1471-2121-10-67
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资金
- Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health
Background: Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P-2] is a critically important regulatory phospholipid found in the plasma membrane of all eukaryotic cells. In addition to being a precursor of important second messengers, PtdIns(4,5)P-2 also regulates ion channels and transporters and serves the endocytic machinery by recruiting clathrin adaptor proteins. Visualization of the localization and dynamic changes in PtdIns(4,5)P-2 levels in living cells is critical to understanding the biology of PtdIns(4,5)P-2. This has been mostly achieved with the use of the pleckstrin homology (PH) domain of PLC delta 1 fused to GFP. Here we report on a comparative analysis of several recently-described yeast PH domains as well as the mammalian Tubby domain to evaluate their usefulness as PtdIns(4,5)P-2 imaging tools. Results: All of the yeast PH domains that have been previously shown to bind PtdIns(4,5)P-2 showed plasma membrane localization but only a subset responded to manipulations of plasma membrane PtdIns(4,5)P-2. None of these domains showed any advantage over the PLC delta 1PH-GFP reporter and were compromised either in their expression levels, nuclear localization or by causing peculiar membrane structures. In contrast, the Tubby domain showed high membrane localization consistent with PtdIns(4,5)P-2 binding and displayed no affinity for the soluble headgroup, Ins(1,4,5)P-3. Detailed comparison of the Tubby and PLC delta 1PH domains showed that the Tubby domain has a higher affinity for membrane PtdIns(4,5)P-2 and therefore displays a lower sensitivity to report on changes of this lipid during phospholipase C activation. Conclusion: These results showed that both the PLC delta 1PH-GFP and the GFP-Tubby domain are useful reporters of PtdIns(4,5)P-2 changes in the plasma membrane, with distinct advantages and disadvantages. While the PLC delta 1PH-GFP is a more sensitive reporter, its Ins(1,4,5)P-3 binding may compromise its accuracy to measure PtdIns(4,5)P-2 changes. The Tubby domain is more accurate to report on PtdIns(4,5)P-2 but its higher affinity and lower sensitivity may limit its utility when phospholipase C activation is only moderate. These studies also demonstrated that similar changes in PtdIns(4,5)P-2 levels in the plasma membrane can differentially regulate multiple effectors if they display different affinities to PtdIns(4,5)P-2.
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