PIP2 Levels Research Articles

Plasma membrane phosphoinositide balance regulates cell shape during Drosophila embryo morphogenesis.

Remodeling of cell shape during morphogenesis is driven by the coordinated expansion and contraction of specific plasma membrane domains. Loss of this coordination results in abnormal cell shape and embryonic lethality. Here, we show that plasma membrane lipid composition plays a key role in coordinating plasma membrane contraction during expansion. We found that an increase in PI(4,5)P2 levels caused premature actomyosin contraction, resulting in the formation of shortened cells. Conversely, acute depletion of PI(4,5)P2 blocked plasma membrane expansion and led to premature actomyosin disassembly. PI(4,5)P2-mediated contractility is counteracted by PI(3,4,5)P3 and the zygotic gene bottleneck, which acts by limiting myosin recruitment during plasma membrane expansion. Collectively, these data support a model in which the ratio of PI(4,5)P2/PI(3,4,5)P3 coordinates actomyosin contractility and plasma membrane expansion during tissue morphogenesis, thus ensuring proper cell shape.

Blockade of the PI-3K signalling pathway by theAggregatibacter actinomycetemcomitanscytolethal distending toxin induces macrophages to synthesize and secrete pro-inflammatory cytokines

The Aggregatibactor actinomycetemcomitans cytolethal distending toxin (Cdt) induces G2 arrest and apoptosis in lymphocytes; these toxic effects are due to the active subunit, CdtB, which functions as a phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase. We now extend our investigation and demonstrate that Cdt is able to perturb human macrophage function. THP-1- and monocyte-derived macrophages were found not to be susceptible to Cdt-induced apoptosis. Nonetheless, the toxin was capable of binding to macrophages and perturbing PI-3K signalling resulting in decreased PIP3 levels and reduced phosphorylation of Akt and GSK3β; these changes were accompanied by concomitant alterations in kinase activity. Exposure of monocytes and macrophages to Cdt resulted in pro-inflammatory cytokine production including increased expression and release of IL-1β, TNFα and IL-6. Furthermore, treatment of cells with either TLR-2, -3 or -4 agonists in the presence of Cdt resulted in an augmented pro-inflammatory response relative to agonist alone. GSK3β inhibitors blocked the Cdt-induced pro-inflammatory cytokine response suggesting a pivotal role for PI-3K blockade, concomitant decrease in GSK3β phosphorylation and increased kinase activity. Collectively, these studies provide new insight into the virulence potential of Cdt in mediating the pathogenesis of disease caused by Cdt-producing organisms.

The lipid kinase PIP5K1C regulates pain signaling and sensitization.

Numerous pain-producing (pronociceptive) receptors signal via phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis. However, it is currently unknown which lipid kinases generate PIP2 in nociceptive dorsal root ganglia (DRG) neurons and if these kinases regulate pronociceptive receptor signaling. Here, we found that phosphatidylinositol 4-phosphate 5 kinase type 1C (PIP5K1C) is expressed at higher levels than any other PIP5K and, based on experiments with Pip5k1c(+/-) mice, generates at least half of all PIP2 in DRG neurons. Additionally, Pip5k1c haploinsufficiency reduces pronociceptive receptor signaling and TRPV1 sensitization in DRG neurons as well as thermal and mechanical hypersensitivity in mouse models of chronic pain. We identified a small molecule inhibitor of PIP5K1C (UNC3230) in a high-throughput screen. UNC3230 lowered PIP2 levels in DRG neurons and attenuated hypersensitivity when administered intrathecally or into the hindpaw. Our studies reveal that PIP5K1C regulates PIP2-dependent nociceptive signaling and suggest that PIP5K1C is a therapeutic target for chronic pain.

Down regulation of brain cellular prion protein in an animal model of insulin resistance: Possible implication in increased prevalence of stroke in pre-diabetics/diabetics

The risk of stroke is drastically increased in diabetic and pre-diabetic patients. The worldwide spread of obesity and insulin resistance increases the incidence of stroke. The direct effect of insulin resistance, as it pertains to stroke, on the central nervous system is not well understood. Since one of the physiological functions of the cellular prion protein (PrPC) is neuroprotection, we studied effects of brain insulin resistance on the expression of PrPC in fructose-fed rats as an animal model of prediabetes. Compared with control chow-fed animals, rats fed a high-fructose diet (FF), exhibited compromised tyrosine phosphorylation of insulin receptor β subunit (IRβ) and reduced serine phosphorylation of Akt, PI3K activity, and decreased PIP3 levels in cortices indicating the induction of brain insulin resistance. We also observed that both mRNA and protein expression of the PrPC was significantly decreased whereas protein level of NR2B subunit of NMDA glutamate receptors profoundly increased in the brain of fructose-fed rats compared to control chow-fed rats. Considering a neuroprotective role for PrPC and the involvement of NR2B subunit in the excitotoxicity-induced neuronal apoptosis, these phenomena may contribute to the severity and poor prognosis of ischemic stroke in diabetes/prediabetes.

PI(4,5)P2 Produced by the PI4P5K SKTL Controls Apical Size by Tethering PAR-3 in Drosophila Epithelial Cells

The control of apical-basal polarity in epithelial layers is a fundamental event in many processes, ranging from embryonic development to tumor formation. A key feature of polarized epithelial cells is their ability to maintain an asymmetric distribution of specific molecular complexes, including the phosphoinositides PI(4,5)P2 and PI(3,4,5)P3. The spatiotemporal regulation of these phosphoinositides iscontrolled by the concerted action of phosphoinositide kinases and phosphatases. Using the Drosophila follicular epithelium as a model system invivo, we show here that PI(4,5)P2 is crucial to maintain apical-basal polarity. PI(4,5)P2 is essentially regulated bythe PI4P5 kinase Skittles (SKTL), whereas neither the phosphatase PTEN nor the PI(4,5)P3 kinase DP110 lead to loss of apical-basal polarity. By inactivating SKTL and thereby strongly reducing PI(4,5)P2 levels in a single cell of the epithelium, we observe the disassembly of adherens junctions, actin cytoskeleton reorganization, and apical constriction leading to delamination, a process similar to that observed during epithelial-mesenchymal transition. We provide evidence that PI(4,5)P2 controls the apical targeting of PAR-3/Bazooka to the plasma membrane and that the loss of this polarized distribution is sufficient to induce a similar cell shape change. Finally, we show that PI(4,5)P2 is excluded from the cell apex and that PAR-3 diffuses laterally just prior to the apical constriction in acontext of endogenous invagination. All together, these results indicate that the PIP5 kinase SKTL, by controlling PI(4,5)P2 polarity, regulates PAR-3 localization and thus the size of the apical domain.

The tumor suppressor PTEN interacts with p53 in hereditary cancer

Numerous hereditary syndromes caused by mutations in multiple tumor suppressor genes can cause cancers. Germline mutations in PTEN and p53 tumor suppressor cause Cowden syndrome and Li-Fraumeni syndrome, respectively. There exists some phenotypic overlap in these syndromes, and they are associated with high risks of breast cancer. The tumor suppressor protein PTEN is a dual-specificity phosphatase which has protein phosphatase activity and lipid phosphatase activity that antagonizes PI3K activity. Cells that lack PTEN have constitutively higher levels of PIP3 and activated downstream targets. PTEN gene is recognized as one of the most frequently mutated or mutated in many human cancers. Li-Fraumeni syndrome results from germline mutations of the tumor suppressor p53 gene encoding a transcriptional factor able to regulate cell cycle and apoptosis when DNA damage occurs. The p53 protein cooperates with PTEN and might be an essential blockage in development of mammary tumors. Many findings have demonstrated that PTEN as well as p53 plays a critical role in DNA damage response. This review summarizes the function of PTEN and p53 in carcinogenic cell signaling. In addition, we will discuss the role of PTEN signaling through its interaction with p53 and MDM2 pathways for the potential implications in hereditary cancer prevention and therapeutic intervention.

TLR4 signaling regulates PI3 kinase activation and macrophage exophagy (796.16)

We have characterized a novel process that macrophages use to degrade species such as aggregated lipoproteins (agLDL) that are too large to be internalized via conventional phagocytosis. Macrophages form an intimate interaction with agLDL through local actin polymerization and form an extracellular compartment to which hydrolytic enzymes are delivered through lysosome exocytosis. The process of extracellular macrophage degradation of large species prior to internalization has been termed “exophagy”. AgLDL degradation by macrophages leads to foam cell formation, a key feature of atherosclerotic plaque development. Therefore unraveling the signaling mechanisms involved in this process may allow therapeutic intervention.Upon engagement with agLDL, Toll‐like receptor 4 (TLR) interacts with its effector MyD88 and TLR4/MyD88 are critical for lysosome exocytosis and actin polymerization during exophagy. At contact sites with agLDL, PI(3,4,5)P3 is produced, suggestive of class I PI3 kinase activation. Inhibition of PI3 kinase impairs lysosome exocytosis and actin polymerization during exophagy. PI(3,4,5)P3 recruits and activates plekstrin homology (PH) domain containing proteins such as protein kinase B (Akt). AgLDL treatment stimulates PI3 kinase dependent Akt activation, and inhibition of Akt using Akti1/2 impairs lysosome exocytosis and actin polymerization. While PI(3,4,5)P3 levels at contact sites with agLDL peak rapidly (around 20‐30 min) and then decline, the sustained generation of PI(4,5)P2 may act as a lasting signal to promote actin polymerization and lysosome exocytosis at sites of exophagy.Exophagy may represent a general mechanism, which cells may use to degrade large extracellular species, and there exists a close relationship between this process and various human diseases. Therefore elucidation of signaling mediators regulating these processes would potentially be of widespread importance in a number of diseases other than atherosclerosis.Grant Funding Source: Supported by NIH

A Long QT Mutation Substitutes Cholesterol for Phosphatidylinositol-4,5-Bisphosphate in KCNQ1 Channel Regulation

IntroductionPhosphatidylinositol-4,5-bisphosphate (PIP2) is a cofactor necessary for the activity of KCNQ1 channels. Some Long QT mutations of KCNQ1, including R243H, R539W and R555C have been shown to decrease KCNQ1 interaction with PIP2. A previous study suggested that R539W is paradoxically less sensitive to intracellular magnesium inhibition than the WT channel, despite a decreased interaction with PIP2. In the present study, we confirm this peculiar behavior of R539W and suggest a molecular mechanism underlying it.Methods and ResultsCOS-7 cells were transfected with WT or mutated KCNE1-KCNQ1 channel, and patch-clamp recordings were performed in giant-patch, permeabilized-patch or ruptured-patch configuration. Similar to other channels with a decreased PIP2 affinity, we observed that the R243H and R555C mutations lead to an accelerated current rundown when membrane PIP2 levels are decreasing. As opposed to R243H and R555C mutants, R539W is not more but rather less sensitive to PIP2 decrease than the WT channel. A molecular model of a fragment of the KCNQ1 C-terminus and the membrane bilayer suggested that a potential novel interaction of R539W with cholesterol stabilizes the channel opening and hence prevents rundown upon PIP2 depletion. We then carried out the same rundown experiments under cholesterol depletion and observed an accelerated R539W rundown that is consistent with this model.ConclusionsWe show for the first time that a mutation may shift the channel interaction with PIP2 to a preference for cholesterol. This de novo interaction wanes the sensitivity to PIP2 variations, showing that a mutated channel with a decreased affinity to PIP2 could paradoxically present a slowed current rundown compared to the WT channel. This suggests that caution is required when using measurements of current rundown as an indicator to compare WT and mutant channel PIP2 sensitivity.

Phosphoinositide Kinases Play Key Roles in Norepinephrine- and Angiotensin II-induced Increase in Phosphatidylinositol 4,5-Bisphosphate and Modulation of Cardiac Function

The seemly paradoxical Gq agonist-stimulated phosphoinositide production has long been known, but the underlying mechanism and its physiological significance are not known. In this study, we studied cardiac phosphoinositide levels in both cells and whole animals under the stimulation of norepinephrine (NE), angiotensin II (Ang II), and other physiologically relevant interventions. The results demonstrated that activation of membrane receptors related to NE or Ang II caused an initial increase and a later fall in phosphatidylinositol 4,5-bisphosphate (PIP2) levels in the primary cultured cardiomyocytes from adult rats. The possible mechanism underlying this increase in PIP2 was found to be through an enhanced activity of phosphatidylinositol 4-kinase IIIβ, which was mediated by an up-regulated interaction between phosphatidylinositol 4-kinase IIIβ and PKC; the increased activity of phosphatidylinositol 4-phosphate 5-kinase γ was also involved for NE-induced increase of PIP2. When the systolic functions of the NE/Ang II-treated cells were measured, a maintained or failed contractility was found to be correlated with a rise or fall in corresponding PIP2 levels. In two animal models of cardiac hypertrophy, PIP2 levels were significantly reduced in hypertrophic hearts induced by isoprenaline but not in those induced by swimming exercise. This study describes a novel mechanism for phosphoinositide metabolism and modulation of cardiac function.

The signaling lipid PI(3,5)P2stabilizes V1–Vosector interactions and activates the V-ATPase

Vacuolar proton-translocating ATPases (V-ATPases) are highly conserved, ATP-driven proton pumps regulated by reversible dissociation of its cytosolic, peripheral V1 domain from the integral membrane V(o) domain. Multiple stresses induce changes in V1-V(o) assembly, but the signaling mechanisms behind these changes are not understood. Here we show that certain stress-responsive changes in V-ATPase activity and assembly require the signaling lipid phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). V-ATPase activation through V1-V(o) assembly in response to salt stress is strongly dependent on PI(3,5)P2 synthesis. Purified V(o) complexes preferentially bind to PI(3,5)P2 on lipid arrays, suggesting direct binding between the lipid and the membrane sector of the V-ATPase. Increasing PI(3,5)P2 levels in vivo recruits the N-terminal domain of V(o)-sector subunit Vph1p from cytosol to membranes, independent of other subunits. This Vph1p domain is critical for V1-V(o) interaction, suggesting that interaction of Vph1p with PI(3,5)P2-containing membranes stabilizes V1-V(o) assembly and thus increases V-ATPase activity. These results help explain the previously described vacuolar acidification defect in yeast fab1 and vac14 mutants and suggest that human disease phenotypes associated with PI(3,5)P2 loss may arise from compromised V-ATPase stability and regulation.

A PIP5 Kinase Essential for Efficient Chemotactic Signaling

SummaryIn neutrophils and Dictyostelium, chemoattractant gradients generate directed cell migration by eliciting signaling events that bias intrinsic motility and favor the production and retention of upgradient pseudopods [1, 2]. Phosphoinositides are actively regulated during chemotaxis in these cells, most iconically in the production of PI(3,4,5)P3 gradients within the plasma membrane [3, 4]. Although it is now known that PI(3,4,5)P3 signaling is nonessential for gradient sensing [5, 6], the role of the related phosphoinositide PI(4,5)P2 is little understood, despite its clear importance in many cell biological processes [7]. We describe here a PIP5 kinase, PikI, which produces PI(4,5)P2 and is essential for efficient chemotaxis of Dictyostelium cells. Without PikI, PI(4,5)P2 levels are reduced by 90%, and while pikI− cells move at normal speeds, they are highly disorientated in cAMP gradients. Following chemotactic stimulation, Ras is efficiently activated in pikI− cells, yet Ras-dependent responses (including activation of PKB) are severely impaired. PikI is phosphorylated by PKB [8], and in vitro studies of a phosphomimic mutant suggest that this phosphorylation increases PikI activity. We propose that adequate PI(4,5)P2 levels are required to couple activated Ras to its downstream effectors and that these levels are regulated by PikI, making it a crucial player in gradient sensing.

PTEN counteracts PIP3 upregulation in spines during NMDA-receptor-dependent long-term depression.

Phosphoinositide 3-kinase (PI3K) and PTEN have been shown to participate in synaptic plasticity during long-term potentiation (LTP) and long-term depression (LTD), respectively. Nevertheless, the dynamics of phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) and the regulation of its synthesis and degradation at synaptic compartments is far from clear. Here, we have used fluorescence resonance energy transfer (FRET) imaging to monitor changes in PIP3 levels in dendritic spines from CA1 hippocampal neurons under basal conditions and upon induction of NMDA receptor (NMDAR)-dependent LTD and LTP. We found that PIP3 undergoes constant turnover in dendritic spines. Contrary to expectations, both LTD and LTP induction trigger an increase in PIP3 synthesis, which requires NMDARs and PI3K activity. Using biochemical methods, the upregulation of PIP3 levels during LTP was estimated to be twofold. However, in the case of LTD, PTEN activity counteracts the increase in PIP3 synthesis, resulting in no net change in PIP3 levels. Therefore, both LTP and LTD signaling converge towards PIP3 upregulation, but PTEN acts as an LTD-selective switch that determines the outcome of PIP3 accumulation.

In KCNQ1 Channels, a Long QT Mutation Induces a Regulation by Cholesterol Instead of Phosphatidylinositol-4,5-Bisphosphate

Phosphatidylinositol-4,5-bisphosphate (PIP2) is a membrane cofactor necessary for the activity of KCNQ1 channels. Some Long QT mutations of KCNQ1, including R243H, R539W and R555C have been shown to decrease KCNQ1 interaction with PIP2. A previous study suggested that when compared to WT channel, R539W is paradoxically less sensitive to PIP2 screening by intracellular magnesium, despite a decreased interaction with PIP2. In the present study, we confirm this peculiar behavior of R539W and suggest a molecular mechanism underlying it. COS-7 cells were transfected with WT or mutated KCNQ1 channel, and patch-clamp recordings in giant-patch or whole-cell configuration were performed. Similar to other channels with a decreased PIP2 affinity, we observed that the R243H and R555C mutations lead to an accelerated current rundown when available PIP2 level is decreased. As opposed to R243H and R555C mutant channels, R539W is not more but rather less sensitive to PIP2 decrease than the WT channel. Consistent with that, the R539W channel is also insensitive to variations in extracellular osmolarity, known to modulate the channel activity via available PIP2. These results suggest that KCNQ1-R539W mutation shortcuts PIP2 in the channel open pore stabilization. Both structural model prediction and functional analysis implicate membrane cholesterol in this effect. This de novo interaction wanes the sensitivity to PIP2 variations, showing that a mutated channel with a decreased affinity to PIP2 could paradoxically present a slowed current rundown compared to the WT channel. This suggests that caution is required when using measurements of current rundown as an indicator to compare WT and mutant channel PIP2 sensitivity.

Membrane Phosphoinositide Turnover by Voltage Sensing Phosphatases

The PI levels can be controlled by the state of dynamic equilibrium between lipid kinase-induced phosphorylation and lipid phosphatase-induced dephosphorylation. The voltage sensing phosphatases (VSPs) gain enzymatic activity upon membrane depolarization so they can trigger temporary imbalance of PIs by electrophysiological method. Recent studies reported that VSPs have both 3- and 5- phosphatase activities upon membrane depolarization. However, its enzymatic characteristics and mechanism are still unclear. Here we examined the functional role of Dr-VSP and Ci-VSP fused with PTEN (Ci-VSPTEN) in the PI turnover through Fluorescence Resonance Energy Transfer (FRET) using Pleckstrin Homology (PH) domains. VSP-induced turnover of diverse PIs, such as PI(3,4)P2, PI(4,5)P2 and PI(3,4,5)P3 showed different kinetics in degradation and resynthesis. When the Dr-VSP expressing cells were given with a 120 mV/3 s of depolarization, the level of PI(4,5)P2, indicated by PH(PLCδ) FRET fairs, decreased rapidly (τ = 1.18 ± 0.17 sec), while the levels of PI(3,4)P2 and PI(3,4,5)P3, indicated by PH(Akt), decreased slowly (τ = 1.36 ± 0.07 sec) and incompletely. The voltage dependent enzymatic activity of phosphatases was also investigated by addressing ramp depolarization which increases from 0 mV to 120 mV for 20 s (holding potential = −80 mV). PH(PLCδ) FRET started to decrease from the voltage of 33.0 ± 6.9 mV, whereas PH(Akt) FRET began to decrease from 71.0 ± 11 mV. In addition, recovery of PI(4,5)P2 and PI(3,4)P2 /PI(3,4,5)P3 showed distinct phenomena in time constants (τrcv = 7.17 ± 1.46 sec and 57.46 ± 4.78 sec, respectively) and in absolute quantity (92.3 ± 8.0 % and 55.3 ± 2.4 %, respectively). Thus, our results suggest that VSP can act as a dual phosphatase and deplete PIs with different time constants. The data also provide the molecular properties of endogenous PI re-synthesis in living cells.

Inhibition by Reduction of PIP2 Accelerates Inactivation of Receptor-Operated TRPC6/7 Currents

TRPC3, C6 and C7, which comprise a subfamily of the mammalian diacylglycerol (DAG)-activated TRPC cation channels, play key roles in cardiovascular, gastrointestinal, renal and nervous system physiology. We recently reported that the depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) dominantly inhibits these TRPC channels activity, irrespective of the presence of a DAG analogue. However, the functional significance of PI(4,5)P2 reduction/depletion during G(q) protein-coupled receptor-operated TRPC activation remains largely unknown. To address this question, we simultaneously measured TRPC current kinetics and PI(4,5)P2/DAG dynamics using Forster resonance energy transfer (FRET) at varying degrees of receptor stimulation. Our measurements demonstrated a clear correlation between the time-course of activation/inactivation in TRPC6/7 currents and the kinetics of PI(4,5)P2 reduction. In contrast, the time-course of DAG production is incompatible with TRPC6/7 current inactivation, indicating that reduction of PI(4,5)P2 is primarily responsible for this inhibition. After estimating the functional dissociation constant of PI(4,5)P2 binding to these DAG-sensitive TRPC channels, we used a model simulation to demonstrate the similarity of receptor-operated TRPC6/7 currents in both a HEK293 cell recombinant expression system and aortic smooth muscle cells (A7r5). Our findings demonstrate that G(q) protein-coupled receptor stimulation causes a reduction in PI(4,5)P2 levels that inhibits TRPC6/7 channels by accelerating their inactivation and perhaps limiting their maximum open probability.

New Experimental Trends for Phosphoinositides Research on Ion Transporter/Channel Regulation

Phosphoinositides(4,5)-bisphosphates [PI(4,5)P2] critically controls membrane excitability, the disruption of which leads to pathophysiological states. PI(4,5)P2 plays a primary role in regulating the conduction and gating properties of ion channels/transporters, through electrostatic and hydrophobic interactions that allow direct associations. In recent years, the development of many molecular tools have brought deep insights into the mechanisms underlying PI(4,5)P2-mediated regulation. This review summarizes the methods currently available to manipulate the cell membrane PI(4,5)P2 level including pharmacological interventions as well as newly designed molecular tools. We concisely introduce materials and experimental designs suitable for the study of PI(4,5)P2-mediated regulation of ion-conducting molecules, in order to assist researchers who are interested in this area. It is our further hope that the knowledge introduced in this review will help to promote our understanding about the pathology of diseases such as cardiac arrhythmias, bipolar disorders, and Alzheimer's disease which are somehow associated with a disruption of PI(4,5)P2 metabolism.

Fixation-induced cell blebbing on spread cells inversely correlates with phosphatidylinositol 4,5-bisphosphate level in the plasma membrane

While most attention has been focused on physiologically generated blebs, the molecular mechanisms for fixation-induced cell blebbing are less investigated. We show that protein-fixing (e.g. aldehydes and picric acid) but not lipid-stabilizing (e.g. OsO4 and KMnO4) fixatives induce blebbing on spread cells. We also show that aldehyde fixation may induce the loss or delocalization of phosphatidylinositol 4,5-bisphosphate (PIP2) in the plasma membrane and that the asymmetric distribution of fixation-induced blebs on spread/migrating cells coincides with that of PIP2 on the cells prefixed by lipid-stabilizing fixatives (e.g., OsO4). Moreover, fixation induces blebbing less readily on PIP2-elevated spread cells but more readily on PIP2-lowered or lipid raft-disrupted spread cells. Our data suggest that fixation-induced lowering of PIP2 level at cytoskeleton-attaching membrane sites causes bleb formation via local breakdown of the membrane–cytoskeleton coupling.

PIP2 Modulates T-Tubule Remodeling During Heart Failure by Working as a Binding Substrate for BIN1

The membrane curvature-forming protein BIN1 is known to be involved in T-tubule formation. Phosphatidylinositol-4,5-bisphosphate (PIP2) has been shown to anchor BIN1 and maintain its localization to the sarcolemma. Since heart failure (HF) is characterized by T-tubule remodeling and dyssynchronous Ca2+ release, we hypothesized that PIP2-mediated targeting of BIN1 to sarcolemma is compromised during the development of HF and is responsible for T-tubule remodeling. We developed an in-vitro model using HL1 cells that developed tubule-like extensions during BIN1 overexpression. These exogenous BIN1-induced tubules were continuous with the sarcolemma (RH-237 staining) similar to T-tubules in ventricular myocytes. To further understand if the BIN1/PIP2 complex is disrupted in HF, we depleted PIP2 in HL1 cells by inhibition of PI4K or activation of PLCβ1. We treated transfected HL1 cells with wortmannin, endothelin-1 (Et-1), m-3M3FBS and a combination of Et-1 + m-3M3FBS. Treated cells showed significantly more tubule degeneration compared to non-treated cells and membrane PIP2 levels decreased as measured by transfecting HL1 cells with a fluorescent PIP2 reporter (PLCδ1-PH-GFP construct). To further determine whether depletion of PIP2-mediated T-tubules loss is correlated with defects in Ca2+ cycling, we isolated adult rat ventricular myocytes, treated them with the same PIP2 depletion agents, then stained them with di-4-ANEPPS in order to assess T-tubule organization. All treated cells showed significantly reduced T-tubule organization than control cells. We also measured Ca2+ cycling in these cells during basal (2000ms) and rapid (1000ms) pacing using flou-2AM. Ca2+ release was delayed in certain cellular regions in treated myocytes and rise time, time to peak and decay time were significantly increased at both pacing rates. These results show that depletion of PIP2 can lead to T-tubule disruption and suggests that PIP2 is required for T-tubule maintenance and function.

Genetically encoded fluorescent probe to visualize intracellular phosphatidylinositol 3,5-bisphosphate localization and dynamics

Phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] is a low-abundance phosphoinositide presumed to be localized to endosomes and lysosomes, where it recruits cytoplasmic peripheral proteins and regulates endolysosome-localized membrane channel activity. Cells lacking PI(3,5)P2 exhibit lysosomal trafficking defects, and human mutations in the PI(3,5)P2-metabolizing enzymes cause lysosome-related diseases. The spatial and temporal dynamics of PI(3,5)P2, however, remain unclear due to the lack of a reliable detection method. Of the seven known phosphoinositides, only PI(3,5)P2 binds, in the low nanomolar range, to a cytoplasmic phosphoinositide-interacting domain (ML1N) to activate late endosome and lysosome (LEL)-localized transient receptor potential Mucolipin 1 (TRPML1) channels. Here, we report the generation and characterization of a PI(3,5)P2-specific probe, generated by the fusion of fluorescence tags to the tandem repeats of ML1N. The probe was mainly localized to the membranes of Lamp1-positive compartments, and the localization pattern was dynamically altered by either mutations in the probe, or by genetically or pharmacologically manipulating the cellular levels of PI(3,5)P2. Through the use of time-lapse live-cell imaging, we found that the localization of the PI(3,5)P2 probe was regulated by serum withdrawal/addition, undergoing rapid changes immediately before membrane fusion of two LELs. Our development of a PI(3,5)P2-specific probe may facilitate studies of both intracellular signal transduction and membrane trafficking in the endosomes and lysosomes.

Abstract B186: The unexpected role of p110β in HER2 amplified and PIK3CA mutant breast cancers.

Abstract The PI3K pathway is alterated in up to 70% of breast cancers. Although both isoform-specific and pan-PI3K inhibitors are available, it is still unclear which compounds are more effective among the different subtypes of breast cancer. We performed time course experiments using the selective p110α inhibitor, BYL-719, in PIK3CA mutant and HER2 amplified cell lines in order to test whether p110α inhibition alone was sufficient to abrogate PI3K-AKT signaling. We observed a rebound of PIP3, the PI3K product, 24 hours after treatment in all cell lines. Notably, both PIP3 and p-AKT levels increased after 24 hours in HER2 amplified cells, while the rebound of p-AKT in PIK3CA mutant was minimal and did not correlate with PIP3 levels. Both p110β inhibitor and siRNA againt p110β were effective in blocking the PIP3 rebound observed after 24 hours of treatment with BYL-719 alone. Consistent with this, the combination of p110α and p110β inhibitors reduce cell growth in long term viability assays and induced a greater apoptotic response in comparison to BYL-719 alone. Importantly, p110α and p110β inhibitors in combination induced tumor regression in vivo, while with single agent BYL-719 we observed only tumor stasis. Finally, we demonstrated, both in vitro and in vivo, that PIP3 levels correlate with inhibitor efficacy more accurately than p-AKT in PIK3CA model. Thus, PIP3 may serve as a more effective biomarker than p-AKT levels to predict response to PI3K inhibitors in PIK3CA mutant and HER2 amplified breast cancers. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B186. Citation Format: Carlotta Costa, Hiromichi Ebi, Anthony C. Faber, Madhuri Nishtala, Young Chul Song, Jeffrey A. Engelman. The unexpected role of p110β in HER2 amplified and PIK3CA mutant breast cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B186.