Phosphatidylinositol (PI) is an essential structural component of eukaryotic membranes that also serves as the common precursor for polyphosphoinositide (PPIn) lipids. Despite the recognized importance of PPIn species for signal transduction and membrane homeostasis, there is still a limited understanding of the relationship between PI availability and the turnover of subcellular PPIn pools. To address these shortcomings, we established a molecular toolbox for investigations of PI distribution within intact cells by exploiting the properties of a bacterial enzyme, PI-specific PLC (PI-PLC). Using these tools, we find a minor presence of PI in membranes of the ER, as well as a general enrichment within the cytosolic leaflets of the Golgi complex, peroxisomes, and outer mitochondrial membrane, but only detect very low steady-state levels of PI within the plasma membrane (PM) and endosomes. Kinetic studies also demonstrate the requirement for sustained PI supply from the ER for the maintenance of monophosphorylated PPIn species within the PM, Golgi complex, and endosomal compartments.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) is a critically important regulatory lipid of the plasma membrane (PM); however, little is known about how cells regulate PM PI(4,5)P 2 levels. Here, we show that the phosphatidylinositol 4-phosphate (PI4P)/phosphatidylserine (PS) transfer activity of the endoplasmic reticulum (ER)–resident ORP5 and ORP8 is regulated by both PM PI4P and PI(4,5)P 2 . Dynamic control of ORP5/8 recruitment to the PM occurs through interactions with the N-terminal Pleckstrin homology domains and adjacent basic residues of ORP5/8 with both PI4P and PI(4,5)P 2 . Although ORP5 activity requires normal levels of these inositides, ORP8 is called on only when PI(4,5)P 2 levels are increased. Regulation of the ORP5/8 attachment to the PM by both phosphoinositides provides a powerful means to determine the relative flux of PI4P toward the ER for PS transport and Sac1-mediated dephosphorylation and PIP 5-kinase–mediated conversion to PI(4,5)P 2 . Using this rheostat, cells can maintain PI(4,5)P 2 levels by adjusting the availability of PI4P in the PM.

Polyphosphoinositides are an important class of lipid that recruit specific effector proteins to organelle membranes. One member, phosphatidylinositol 4-phosphate (PtdIns4 P ) has been localized to Golgi membranes based on the distribution of lipid binding modules from PtdIns4 P effector proteins. However, these probes may be biased by additional interactions with other Golgi-specific determinants. In this paper, we derive a new PtdIns4 P biosensor using the PtdIns4 P binding of SidM (P4M) domain of the secreted effector protein SidM from the bacterial pathogen Legionella pneumophila . PtdIns4 P was necessary and sufficient for localization of P4M, which revealed pools of the lipid associated not only with the Golgi but also with the plasma membrane and Rab7-positive late endosomes/lysosomes. PtdIns4 P distribution was determined by the localization and activities of both its anabolic and catabolic enzymes. Therefore, P4M reports a wider cellular distribution of PtdIns4 P than previous probes and therefore will be valuable for dissecting the biological functions of PtdIns4 P in its assorted membrane compartments.

Successful completion of cytokinesis relies on addition of new membrane, and requires the recycling endosome regulator Rab11, which localizes to the midzone. Despite the critical role of Rab11 in this process, little is known about the formation and composition of Rab11-containing organelles. Here, we identify the phosphatidylinositol (PI) 4-kinase III β Four wheel drive (Fwd) as a key regulator of Rab11 during cytokinesis in Drosophila melanogaster spermatocytes. We show Fwd is required for synthesis of PI 4-phosphate (PI4P) on Golgi membranes and for formation of PI4P-containing secretory organelles that localize to the midzone. Fwd binds and colocalizes with Rab11 on Golgi membranes, and is required for localization of Rab11 in dividing cells. A kinase-dead version of Fwd also binds Rab11 and partially restores cytokinesis to fwd mutant flies. Moreover, activated Rab11 partially suppresses loss of fwd . Our data suggest Fwd plays catalytic and noncatalytic roles in regulating Rab11 during cytokinesis.

Cell–cell communication through connexin43 (Cx43)-based gap junction channels is rapidly inhibited upon activation of various G protein–coupled receptors; however, the mechanism is unknown. We show that Cx43-based cell–cell communication is inhibited by depletion of phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P 2 ) from the plasma membrane. Knockdown of phospholipase Cβ3 (PLCβ3) inhibits PtdIns(4,5)P 2 hydrolysis and keeps Cx43 channels open after receptor activation. Using a translocatable 5-phosphatase, we show that PtdIns(4,5)P 2 depletion is sufficient to close Cx43 channels. When PtdIns(4,5)P 2 is overproduced by PtdIns(4)P 5-kinase, Cx43 channel closure is impaired. We find that the Cx43 binding partner zona occludens 1 (ZO-1) interacts with PLCβ3 via its third PDZ domain. ZO-1 is essential for PtdIns(4,5)P 2 -hydrolyzing receptors to inhibit cell–cell communication, but not for receptor–PLC coupling. Our results show that PtdIns(4,5)P 2 is a key regulator of Cx43 channel function, with no role for other second messengers, and suggest that ZO-1 assembles PLCβ3 and Cx43 into a signaling complex to allow regulation of cell–cell communication by localized changes in PtdIns(4,5)P 2 .

Rapamycin (rapa)-induced heterodimerization of the FRB domain of the mammalian target of rapa and FKBP12 was used to translocate a phosphoinositide 5-phosphatase (5-ptase) enzyme to the plasma membrane (PM) to evoke rapid changes in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5) P 2 ) levels. Rapa-induced PM recruitment of a truncated type IV 5-ptase containing only the 5-ptase domain fused to FKBP12 rapidly decreased PM PtdIns(4,5) P 2 as monitored by the PLCδ1PH-GFP fusion construct. This decrease was paralleled by rapid termination of the ATP-induced Ca 2+ signal and the prompt inactivation of menthol-activated transient receptor potential melastatin 8 (TRPM8) channels. Depletion of PM PtdIns(4,5) P 2 was associated with a complete blockade of transferrin uptake and inhibition of epidermal growth factor internalization. None of these changes were observed upon rapa-induced translocation of an mRFP-FKBP12 fusion protein that was used as a control. These data demonstrate that rapid inducible depletion of PM PtdIns(4,5) P 2 is a powerful tool to study the multiple regulatory roles of this phospholipid and to study differential sensitivities of various processes to PtdIns(4,5) P 2 depletion.

Agonist-induced endocytosis and processing of the G protein–coupled AT 1 angiotensin II (Ang II) receptor (AT 1 R) was studied in HEK 293 cells expressing green fluorescent protein (GFP)– or hemagglutinin epitope–tagged forms of the receptor. After stimulation with Ang II, the receptor and its ligand colocalized with Rab5–GFP and Rab4–GFP in early endosomes, and subsequently with Rab11–GFP in pericentriolar recycling endosomes. Inhibition of phosphatidylinositol (PI) 3-kinase by wortmannin (WT) or LY294002 caused the formation of large endosomal vesicles of heterogeneous Rab composition, containing the ligand–receptor complex in their limiting membranes and in small associated vesicular structures. In contrast to Alexa ® –transferrin, which was mainly found in small vesicles associated with the outside of large vesicles in WT-treated cells, rhodamine–Ang II was also segregated into small internal vesicles. In cells labeled with 125 I-Ang II, WT treatment did not impair the rate of receptor endocytosis, but significantly reduced the initial phase of receptor recycling without affecting its slow component. Similarly, WT inhibited the early, but not the slow, component of the recovery of AT 1 R at the cell surface after termination of Ang II stimulation. These data indicate that internalized AT 1 receptors are processed via vesicles that resemble multivesicular bodies, and recycle to the cell surface by a rapid PI 3-kinase–dependent recycling route, as well as by a slower pathway that is less sensitive to PI 3-kinase inhibitors.

Phagocytosis is a highly localized and rapid event, requiring the generation of spatially and temporally restricted signals. Because phosphatidylinositol 3-kinase (PI3K) plays an important role in the innate immune response, we studied the generation and distribution of 3′ phosphoinositides (3′PIs) in macrophages during the course of phagocytosis. The presence of 3′PI was monitored noninvasively in cells transfected with chimeras of green fluorescent protein and the pleckstrin homology domain of either Akt, Btk, or Gab1. Although virtually undetectable in unstimulated cells, 3′PI rapidly accumulated at sites of phagocytosis. This accumulation was sharply restricted to the phagosomal cup, with little 3′PI detectable in the immediately adjacent areas of the plasmalemma. Measurements of fluorescence recovery after photobleaching were made to estimate the mobility of lipids in the cytosolic monolayer of the phagosomal membrane. Stimulation of phagocytic receptors induced a marked reduction of lipid mobility that likely contributes to the restricted distribution of 3′PI at the cup. 3′PI accumulation during phagocytosis was transient, terminating shortly after sealing of the phagosomal vacuole. Two factors contribute to the rapid disappearance of 3′PI: the dissociation of the type I PI3K from the phagosomal membrane and the persistent accumulation of phosphoinositide phosphatases.

The carrier-mediated, electroneutral exchange of Na + for H + across the plasma membrane does not directly consume metabolic energy. Nevertheless, acute depletion of cellular ATP markedly decreases transport. We analyzed the possible involvement of polyphosphoinositides in the metabolic regulation of NHE1, the ubiquitous isoform of the Na + /H + exchanger. Depletion of ATP was accompanied by a marked reduction of plasmalemmal phosphatidylinositol 4,5-bisphosphate (PIP 2 ) content. Moreover, sequestration or hydrolysis of plasmalemmal PIP 2 , in the absence of ATP depletion, was associated with profound inhibition of NHE1 activity. Examination of the primary structure of the COOH-terminal domain of NHE1 revealed two potential PIP 2 -binding motifs. Fusion proteins encoding these motifs bound PIP 2 in vitro. When transfected into antiport-deficient cells, mutant forms of NHE1 lacking the putative PIP 2 -binding domains had greatly reduced transport capability, implying that association with PIP 2 is required for optimal activity. These findings suggest that NHE1 activity is modulated by phosphoinositides and that the inhibitory effect of ATP depletion may be attributable, at least in part, to the accompanying net dephosphorylation of PIP 2 .

Phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P 2 ) pools that bind pleckstrin homology (PH) domains were visualized by cellular expression of a phospholipase C (PLC)δ 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 PLCδ PH domain known to form critical contacts with PtdIns(4,5)P 2 . 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 Ca 2+ chelation. In both ionomycin- and agonist-stimulated cells, fluorescent probe distribution closely correlated with changes in absolute mass of PtdIns(4,5)P 2 . Inhibition of PtdIns(4,5)P 2 synthesis by quercetin or phenylarsine oxide prevented the relocalization of the fluorescent probe to the membranes after Ca 2+ chelation in ionomycin-treated cells or during agonist stimulation. In contrast, the synthesis of the PtdIns(4,5)P 2 imaged by the PH domain was not sensitive to concentrations of wortmannin that had been found inhibitory of the synthesis of myo-[ 3 H]inositol– labeled PtdIns(4,5)P 2 . Identification and dynamic imaging of phosphoinositides that interact with PH domains will further our understanding of the regulation of such proteins by inositol phospholipids.