In:
BMC Cell Biology, Springer Science and Business Media LLC, Vol. 10, No. 1 ( 2009-12)
Abstract:
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 PLCδ1 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 PLCδ1PH-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 PLCδ1PH 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 PLCδ1PH-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 PLCδ1PH-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 .
Type of Medium:
Online Resource
ISSN:
1471-2121
DOI:
10.1186/1471-2121-10-67
Language:
English
Publisher:
Springer Science and Business Media LLC
Publication Date:
2009
detail.hit.zdb_id:
2964981-X
detail.hit.zdb_id:
2041486-9
SSG:
12
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