Live cell imaging with protein domains capable of recognizing phosphatidylinositol 4,5-bisphosphate; a comparative study

Abstract

BackgroundPhosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] 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)P2 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)P2 levels in living cells is critical to understanding the biology of PtdIns(4,5)P2. 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)P2 imaging tools.ResultsAll of the yeast PH domains that have been previously shown to bind PtdIns(4,5)P2 showed plasma membrane localization but only a subset responded to manipulations of plasma membrane PtdIns(4,5)P2. 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)P2 binding and displayed no affinity for the soluble headgroup, Ins(1,4,5)P3. Detailed comparison of the Tubby and PLCδ1PH domains showed that the Tubby domain has a higher affinity for membrane PtdIns(4,5)P2 and therefore displays a lower sensitivity to report on changes of this lipid during phospholipase C activation.ConclusionThese results showed that both the PLCδ1PH-GFP and the GFP-Tubby domain are useful reporters of PtdIns(4,5)P2 changes in the plasma membrane, with distinct advantages and disadvantages. While the PLCδ1PH-GFP is a more sensitive reporter, its Ins(1,4,5)P3 binding may compromise its accuracy to measure PtdIns(4,5)P2changes. The Tubby domain is more accurate to report on PtdIns(4,5)P2 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)P2 levels in the plasma membrane can differentially regulate multiple effectors if they display different affinities to PtdIns(4,5)P2.