Trends in Pharmacological Sciences
Current AwarenessHow accurately can we image inositol lipids in living cells?
Section snippets
Use of protein modules that recognize inositol lipids to visualize lipid distribution
The functions of an increasing number of signalling proteins (e.g. ion channels, ion pumps, protein and lipid kinases and phosphatases, and regulators of either GTP-binding proteins or the cytoskeleton) have been shown to be regulated, with remarkable stereospecificity, by inositol lipids. In many of these cases the protein motifs that mediate the interactions with the various inositol lipids have been defined, often with detailed three-dimensional structural information. These domains possess
Can all the cellular lipids be ‘imaged’ with these probes?
Although these studies convincingly demonstrate that the distribution of these fluorescent probes is dependent on the presence or production of a particular inositol lipid, the question has been raised, from the beginning, whether the method allows equal detection of all of the cellular inositides. For example, tagging of the PH domain of PLCδ1 with GFP recognizes plasma membrane PtdIns(4,5)P2 but fails to label intracellular structures (e.g. Golgi, the nuclear envelope or secretory vesicles)
Changes in the levels of soluble inositol phosphates can complicate interpretations
A further issue to be considered when interpreting data on the cellular distribution and dynamics of the PH domains8 is the equal or higher apparent in vitro affinity of these proteins to the soluble inositol phosphate isomers compared with the cognate inositol lipid. A good example is again PLCδ1-PH, which binds Ins(1,4,5)P3 with about 10 times higher affinity than PtdIns(4,5)P2 in vitro. Based on this finding, it has been proposed that translocation of the PLCδ1-PH–GFP protein from the
Additional interactions might complement lipid-binding and determine membrane localization of the fluorescent probes
The interpretation of data obtained by following the distribution of these probes depends on whether the binding to the inositol lipid head group is the sole determinant of their membrane association. A protein might not necessarily localize to the plasma membrane simply because it binds Ins(1,4,5)P3 [or the inositol lipid head group of PtdIns(4,5)P2]. For example, the isolated Ins(1,4,5)P3-binding domain of the Ins(1,4,5)P3 receptor does not localize to the plasma membrane (P. Várnai and T.
Concluding remarks
The reader might be wondering whether this recently developed elegant and promising technique provides more trouble than benefit, but this is certainly not the case. In fact, we strongly believe that the use of these novel tools will permit the exploration of the inositide-regulatory paradigm at a different level and, more importantly, from a new direction. Based on these studies we have realized that not all inositol lipids exist in a ‘free’ form and some must be complexed with proteins.
Acknowledgements
We are most grateful to Kees Jalink and Thomas F. Martin for sharing information on unpublished results, and to Kevin J. Catt for fruitful discussions and critical reading of the manuscript.
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