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J. Cell Biol.,
Volume 143, Number 2, October 19, 1998 501-510
Endocrinology and Reproduction Research Branch, National Institutes of Child Health and Human Development, National
Institutes of Health, Bethesda, Maryland 20892-4510
Phosphatidylinositol 4,5-bisphosphate
(PtdIns[4,5]P2) pools that bind pleckstrin homology
(PH) domains were visualized by cellular expression of
a phospholipase C (PLC)
PH domain-green fluorescent protein fusion construct and analysis of confocal images in living cells. Plasma membrane localization of
the fluorescent probe required the presence of three
basic residues within the PLC PH domain known to
form critical contacts with PtdIns(4,5)P2. Activation of
endogenous PLCs by ionophores or by receptor stimulation produced rapid redistribution of the fluorescent
signal from the membrane to cytosol, which was reversed after Ca2+ chelation. In both ionomycin- and agonist-stimulated cells, fluorescent probe distribution
closely correlated with changes in absolute mass of
PtdIns(4,5)P2. Inhibition of PtdIns(4,5)P2 synthesis by quercetin or phenylarsine oxide prevented the relocalization of the fluorescent probe to the membranes after
Ca2+ chelation in ionomycin-treated cells or during
agonist stimulation. In contrast, the synthesis of the
PtdIns(4,5)P2 imaged by the PH domain was not sensitive to concentrations of wortmannin that had been
found inhibitory of the synthesis of myo-[3H]inositol-
labeled PtdIns(4,5)P2. Identification and dynamic imaging of phosphoinositides that interact with PH domains
will further our understanding of the regulation of such
proteins by inositol phospholipids.
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