Cellular Regulation of Extension and Retraction of Pseudopod-Like Blebs Produced by Nanosecond Pulsed Electric Field (nsPEF)

Recently we described a new phenomenon of anodotropic pseudopod-like blebbing in U937 cells exposed to nanosecond pulsed electric field (nsPEF). In Ca²⁺-free buffer such exposure initiates formation of pseudopod-like blebs (PLBs), protrusive cylindrical cell extensions that are distinct from apoptotic and necrotic blebs. PLBs nucleate predominantly on anode-facing cell pole and extend toward anode during nsPEF exposure. Bleb extension depends on actin polymerization and availability of actin monomers. Inhibition of intracellular Ca²⁺, cell contractility, and RhoA produced no effect on PLB initiation. Meanwhile, inhibition of WASP by wiskostatin causes dose-dependent suppression of PLB growth. Soon after the end of nsPEF exposure PLBs lose directionality of growth and then retract. Microtubule toxins nocodazole and paclitaxel did not show immediate effect on PLBs; however, nocodazole increased mobility of intracellular components during PLB extension and retraction. Retraction of PLBs is produced by myosin activation and the corresponding increase in PLB cortex contractility. Inhibition of myosin by blebbistatin reduces retraction while inhibition of RhoA–ROCK pathway by Y-27632 completely prevents retraction. Contraction of PLBs can produce cell translocation resembling active cell movement. Overall, the formation, properties, and life cycle of PLBs share common features with protrusions associated with ameboid cell migration. PLB life cycle may be controlled through activation of WASP by its upstream effectors such as Cdc42 and PIP2, and main ROCK activator—RhoA. Parallels between pseudopod-like blebbing and motility blebbing may provide new insights into their underlying mechanisms.     

Membrane permeabilization and cell damage by ultrashort electric field shocks

Mammalian cells exposed to electric field pulses of nanosecond duration (nsPEF; 60-ns, 12 kV/cm) experienced a profound and long-lasting increase in passive electrical conductance (G_m) of the cell membrane, probably caused by opening of stable conductance pores (CPs). The CPs were permeable to Cl⁻ and alkali metal cations, but not to larger molecules such as propidium iodide (PI). CPs gradually resealed; the process took minutes and could be observed even in dialyzed cells and in ATP- and glucose-free solutions. Cells subjected to long nsPEF trains (up to 200 pulses) underwent severe and immediate necrotic transformation (cell swelling, blebbing, cytoplasm granulation), but remained impermeable to PI for at least 30-60 min after the exposure. Both G_m increase after short nsPEF trains and necrotic changes after long nsPEF trains were cell type-dependent: they were much weaker in HeLa than in GH3 cells. La³⁺ and Gd³⁺ ions significantly inhibited the nsPEF-induced G_m increase (probably by blocking the CPs), and effectively protected intensely exposed cells from developing necrosis. We conclude that plasma membrane permeabilization is the principal cause of necrotic transformation in nsPEF-exposed cells and probably contributes to other known nsPEF bioeffects.     

Exchange Factor EFA6R Requires C-terminal Targeting to the Plasma Membrane to Promote Cytoskeletal Rearrangement through the Activation of ADP-ribosylation Factor 6 (ARF6)

ADP-ribosylation factor 6 (ARF6) small GTPase regulates membrane trafficking and cytoskeleton rearrangements at the plasma membrane (PM) by cycling between the GTP-bound active and GDP-bound inactive conformations. Guanine nucleotide exchange factors (GEFs) activate ARF6. The exchange factor for ARF6 (EFA6) R has been identified as a biomarker for ovarian cancer. EFA6R shares the catalytic Sec7, pleckstrin homology (PH), and coiled coil (CC) domains of the other EFA6 family GEFs. Here we report the functional characterization of EFA6R. Endogenous EFA6R was present in the plasma membrane fraction. The exogenously expressed FLAG- and GFP-tagged EFA6R were targeted to the PM. In vitro, GFP-EFA6R associated weakly but preferentially with phosphatidylinositol 4,5-bisphosphate (PIP₂) through the PH domain. EFA6R required both its PH and CC domains localized at the C terminus to target the PM. Consistent with this, EFA6R lacking the CC domain (EFA6RΔCC) was released from the PM into the cytosol upon PIP₂ depletion, whereas EFA6R release from the PM required both PIP₂ depletion and actin destabilization. These results suggest that the dual targeting via the PH and CC domains is important for the PM localization of EFA6R. EFA6R specifically catalyzed the GTP loading of ARF6 in mammalian cells. Moreover, EFA6R regulated ARF6 localization and thereby actin stress fiber loss. The GEF activity of EFA6R was dependent on the presence of the Sec7 domain. The PH and CC domains were also required for the in vivo GEF activity of EFA6R but could be functionally replaced by the CAAX motif of K-Ras, suggesting a role for these domains in the membrane targeting of EFA6R.     

Phosphatidylinositol 4,5-Bisphosphate Homeostasis Regulated by Nir2 and Nir3 Proteins at Endoplasmic Reticulum-Plasma Membrane Junctions

Phosphatidylinositol (PI) 4,5-bisphosphate (PIP₂) at the plasma membrane (PM) constitutively controls many cellular functions, and its hydrolysis via receptor stimulation governs cell signaling. The PI transfer protein Nir2 is essential for replenishing PM PIP₂ following receptor-induced hydrolysis, but key mechanistic aspects of this process remain elusive. Here, we demonstrate that PI at the membrane of the endoplasmic reticulum (ER) is required for the rapid replenishment of PM PIP₂ mediated by Nir2. Nir2 detects PIP₂ hydrolysis and translocates to ER-PM junctions via binding to phosphatidic acid. With distinct phosphatidic acid binding abilities and PI transfer protein activities, Nir2 and its homolog Nir3 differentially regulate PIP₂ homeostasis in cells during intense receptor stimulation and in the resting state, respectively. Our study reveals that Nir2 and Nir3 work in tandem to achieve different levels of feedback based on the consumption of PM PIP₂ and function at ER-PM junctions to mediate nonvesicular lipid transport between the ER and the PM.     

The Cytoskeleton As A Target In Quinone Toxicity

The exposure of mammalian cells to toxic concentrations of redox cycling and alkylating quinones causes marked changes in cell surface structure knoun as plasma membrane blebbing. These alterations are associated with the redistribution of plasma membrane proteins and the disruption of the normal organization of the cytoskeletal microfilaments which appears 10 be due mainly to actin cross-linking and dissociation of α-actinin from the actin network. The major hiochemical mechanisms responsible for these effects seem to involve the depletion of cytoskeletal protein dfhydryl groups and the increase cytosolic Ca²⁺ concentration following the alkylation/oxidation of free sulfhydryl groups in several Ca²⁺ transport systems. Depletion of intracellular ATP is also associated with quinone-induced plasma menibrane blebbing. However, ATP depletion occurs well after the onset of the morphological changes. and thus it does not seem to be causatively related to their appeardncc Thiol reductants. such as dithiothreitol. efficiently prevent the oxidation of cytoskeletal protein thiols. the increase in cytosolic free Ca²⁺ concentration and cell blebbing induced by redox cycling. but not alkylating. quinones. These results demonstrate that alkylating and redox cycling quinones cause siinilar structural and biochemical modifications of the cytoskeleton by means of different mechanisms. namely alkylation and oxidation of critical sulfhydryl groups     

Open Conformation of Ezrin Bound to Phosphatidylinositol 4,5-Bisphosphate and to F-actin Revealed by Neutron Scattering

Ezrin is a member of the ezrin-radixin-moesin family (ERM) of adapter proteins that are localized at the interface between the cell membrane and the cortical actin cytoskeleton, and they regulate a variety of cellular functions. The structure representing a dormant and closed conformation of an ERM protein has previously been determined by x-ray crystallography. Here, using contrast variation small angle neutron scattering, we reveal the structural changes of the full-length ezrin upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP₂) and to F-actin. Ezrin binding to F-actin requires the simultaneous binding of ezrin to PIP₂. Once bound to F-actin, the opened ezrin forms more extensive contacts with F-actin than generally depicted, suggesting a possible role of ezrin in regulating the interfacial structure and dynamics between the cell membrane and the underlying actin cytoskeleton. In addition, using gel filtration, we find that the conformational opening of ezrin in response to PIP₂ binding is cooperative, but the cooperativity is disrupted by a phospho-mimic mutation S249D in the 4.1-ezrin/radixin/moesin (FERM) domain of ezrin. Using surface plasmon resonance, we show that the S249D mutation weakens the binding affinity and changes the kinetics of 4.1-ERM to PIP₂ binding. The study provides the first structural view of the activated ezrin bound to PIP₂ and to F-actin.     

Nir1 constitutively localizes at ER–PM junctions and promotes Nir2 recruitment for PIP2 homeostasis

Homeostatic regulation of plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate (PIP₂) in receptor-stimulated cells is mediated by the lipid transfer protein Nir2. Nir2 is dynamically recruited to endoplasmic reticulum–plasma membrane (ER–PM) junctions to facilitate replenishment of PM PIP₂ hydrolyzed during receptor-mediated signaling. However, our knowledge regarding the activation and sustainment of Nir2-mediated replenishment of PM PIP₂ is limited. Here, we describe the functions of Nir1 as a positive regulator of Nir2 and PIP₂ homeostasis. In contrast to the family proteins Nir2 and Nir3, Nir1 constitutively localizes at ER–PM junctions. Nir1 potentiates Nir2 targeting to ER–PM junctions during receptor-mediated signaling and is required for efficient PM PIP₂ replenishment. Live-cell imaging and biochemical analysis reveal that Nir1 interacts with Nir2 via a region between the FFAT motif and the DDHD domain. Combined, results from this study identify Nir1 as an ER–PM junction localized protein that promotes Nir2 recruitment for PIP₂ homeostasis.     

Bnip3 and AIF cooperate to induce apoptosis and cavitation during epithelial morphogenesis

Increasing evidence supports a critical role for the septin cytoskeleton at the plasma membrane during physiological processes including motility, formation of dendritic spines or cilia, and phagocytosis. We sought to determine how septins regulate the plasma membrane, focusing on this cytoskeletal element's role during effective amoeboid motility. Surprisingly, septins play a reactive rather than proactive role, as demonstrated during the response to increasing hydrostatic pressure and subsequent regulatory volume decrease. In these settings, septins were required for rapid cortical contraction, and SEPT6-GFP was recruited into filaments and circular patches during global cortical contraction and also specifically during actin filament depletion. Recruitment of septins was also evident during excessive blebbing initiated by blocking membrane trafficking with a dynamin inhibitor, providing further evidence that septins are recruited to facilitate retraction of membranes during dynamic shape change. This function of septins in assembling on an unstable cortex and retracting aberrantly protruding membranes explains the excessive blebbing and protrusion observed in septin-deficient T cells.     

Thresholds for Phosphatidylserine Externalization in Chinese Hamster Ovarian Cells following Exposure to Nanosecond Pulsed Electrical Fields (nsPEF)

High-amplitude, MV/m, nanosecond pulsed electric fields (nsPEF) have been hypothesized to cause nanoporation of the plasma membrane. Phosphatidylserine (PS) externalization has been observed on the outer leaflet of the membrane shortly after nsPEF exposure, suggesting local structural changes in the membrane. In this study, we utilized fluorescently-tagged Annexin V to observe the externalization of PS on the plasma membrane of isolated Chinese Hamster Ovary (CHO) cells following exposure to nsPEF. A series of experiments were performed to determine the dosimetric trends of PS expression caused by nsPEF as a function of pulse duration, τ, delivered field strength, E_D, and pulse number, n. To accurately estimate dose thresholds for cellular response, data were reduced to a set of binary responses and ED50s were estimated using Probit analysis. Probit analysis results revealed that PS externalization followed the non-linear trend of (τ*E_D ²)⁻¹ for high amplitudes, but failed to predict low amplitude responses. A second set of experiments was performed to determine the nsPEF parameters necessary to cause observable calcium uptake, using cells preloaded with calcium green (CaGr), and membrane permeability, using FM1-43 dye. Calcium influx and FM1-43 uptake were found to always be observed at lower nsPEF exposure parameters compared to PS externalization. These findings suggest that multiple, higher amplitude and longer pulse exposures may generate pores of larger diameter enabling lateral diffusion of PS; whereas, smaller pores induced by fewer, lower amplitude and short pulse width exposures may only allow extracellular calcium and FM1-43 uptake.     

Mechanism of PLC-Mediated Kir3 Current Inhibition

A large number of ion channels maintain their activity through direct interactions with phosphatidylinositol bisphosphate (PIP₂). For such channels, hydrolysis of PIP₂ causes current inhibition. It has become controversial whether the inhibitory effects on channel activity represent direct effects of PIP₂ hydrolysis or of downstream PKC action. We studied Phospholipase C (PLC)-dependent inhibition of G protein-activated inwardly rectifying K⁺ (Kir3) channels. By monitoring simultaneously channel activity and PIP2 hydrolysis, we determined that both direct PIP₂ depletion and PKC actions contribute to Kir3 current inhibition. We show that the PKC-induced effects strongly depend on the PIP₂ levels in the membrane. At the same time, we show that PKC destabilizes Kir3/PIP₂ interactions and enhances the effects of PIP₂ depletion on channel activity. These results demonstrate that PIP₂ depletion and PKC-mediated effects reinforce each other and suggest that both of these interdependent mechanisms contribute to Kir3 current inhibition. This mechanistic insight may explain how even minor changes in PIP₂ levels can have profound effects on Kir3 activity. We also show that stabilization of Kir3PIP₂ interactions with Gβγ attenuates both PKC and Gq-mediated current inhibition, suggesting that diverse pathways regulate Kir3 activity through modulation of channel interactions with PIP₂.     

NuMA interacts with phosphoinositides and links the mitotic spindle with the plasma membrane

The positioning and the elongation of the mitotic spindle must be carefully regulated. In human cells, the evolutionary conserved proteins LGN/Gα_i1‐3 anchor the coiled‐coil protein NuMA and dynein to the cell cortex during metaphase, thus ensuring proper spindle positioning. The mechanisms governing cortical localization of NuMA and dynein during anaphase remain more elusive. Here, we report that LGN/Gα_i1‐3 are dispensable for NuMA‐dependent cortical dynein enrichment during anaphase. We further establish that NuMA is excluded from the equatorial region of the cell cortex in a manner that depends on the centralspindlin components CYK4 and MKLP1. Importantly, we reveal that NuMA can directly associate with PtdInsP (PIP) and PtdInsP₂ (PIP₂) phosphoinositides in vitro. Furthermore, chemical or enzymatic depletion of PIP/PIP₂ prevents NuMA cortical localization during mitosis, and conversely, increasing PIP₂ levels augments mitotic cortical NuMA. Overall, our study uncovers a novel function for plasma membrane phospholipids in governing cortical NuMA distribution and thus the proper execution of mitosis.     

Dual-mode phospholipid regulation of human inward rectifying potassium channels

The lipid bilayer is a critical determinant of ion channel activity; however, efforts to define the lipid dependence of channel function have generally been limited to cellular expression systems in which the membrane composition cannot be fully controlled. We reconstituted purified human Kir2.1 and Kir2.2 channels into liposomes of defined composition to study their phospholipid dependence of activity using ⁸⁶Rb⁺ flux and patch-clamp assays. Our results demonstrate that Kir2.1 and Kir2.2 have two distinct lipid requirements for activity: a specific requirement for phosphatidylinositol 4,5-bisphosphate (PIP₂) and a nonspecific requirement for anionic phospholipids. Whereas we previously showed that PIP₂ increases the channel open probability, in this work we find that activation by POPG increases both the open probability and unitary conductance. Oleoyl CoA potently inhibits Kir2.1 by antagonizing the specific requirement for PIP₂, and EPC appears to antagonize activation by the nonspecific anionic requirement. Phosphatidylinositol phosphates can act on both lipid requirements, yielding variable and even opposite effects on Kir2.1 activity depending on the lipid background. Mutagenesis experiments point to the role of intracellular residues in activation by both PIP₂ and anionic phospholipids. In conclusion, we utilized purified proteins in defined lipid membranes to quantitatively determine the phospholipid requirements for human Kir channel activity.     

Force Spectroscopy Measurements Show That Cortical Neurons Exposed to Excitotoxic Agonists Stiffen before Showing Evidence of Bleb Damage

In ischemic and traumatic brain injury, hyperactivated glutamate (N-methyl-D-aspartic acid, NMDA) and sodium (Nav) channels trigger excitotoxic neuron death. Na⁺, Ca⁺⁺ and H₂O influx into affected neurons elicits swelling (increased cell volume) and pathological blebbing (disassociation of the plasma membrane’s bilayer from its spectrin-actomyosin matrix). Though usually conflated in injured tissue, cell swelling and blebbing are distinct processes. Around an injury core, salvageable neurons could be mildly swollen without yet having suffered the bleb-type membrane damage that, by rendering channels leaky and pumps dysfunctional, exacerbates the excitotoxic positive feedback spiral. Recognizing when neuronal inflation signifies non-lethal osmotic swelling versus blebbing should further efforts to salvage injury-penumbra neurons. To assess whether the mechanical properties of osmotically-swollen versus excitotoxically-blebbing neurons might be cytomechanically distinguishable, we measured cortical neuron elasticity (gauged via atomic force microscopy (AFM)-based force spectroscopy) upon brief exposure to hypotonicity or to excitotoxic agonists (glutamate and Nav channel activators, NMDA and veratridine). Though unperturbed by solution exchange per se, elasticity increased abruptly with hypotonicity, with NMDA and with veratridine. Neurons then invariably softened towards or below the pre-treatment level, sometimes starting before the washout. The initial channel-mediated stiffening bespeaks an abrupt elevation of hydrostatic pressure linked to NMDA or Nav channel-mediated ion/H₂O fluxes, together with increased [Ca⁺⁺]_int-mediated submembrane actomyosin contractility. The subsequent softening to below-control levels is consistent with the onset of a lethal level of bleb damage. These findings indicate that dissection/identification of molecular events during the excitotoxic transition from stiff/swollen to soft/blebbing is warranted and should be feasible.     

Differential Effects of Lysophosphatidic Acid and Phosphatidylinositol 4,5-Bisphosphate on Actin Dynamics by Direct Association with the Actin-binding Protein Villin

We have previously reported that the epithelial cell-specific actin-binding protein villin directly associates with phosphatidylinositol 4,5-bisphosphate (PIP₂) through three binding sites that overlap with actin-binding sites in villin. As a result, association of villin with PIP₂ in hibits actin depolymerization and enhances actin cross-linking by villin. In this study, we demonstrate that these three PIP₂-binding sites also bind the more hydrophilic phospholipid, lysophosphatidic acid (LPA) but with a higher affinity than PIP₂ (dissociation constant (K_d) of 22 μm versus 39.5 μm for PIP₂). More interestingly, unlike PIP₂, the association of villin with LPA inhibits all actin regulatory functions of villin. In addition, unlike PIP₂, LPA dramatically stimulates the tyrosine phosphorylation of villin by c-Src kinase. These studies suggest that in cells, selective interaction of villin with either PIP₂ or LPA could have dramatically different outcomes on actin reorganization as well as phospholipid-regulated cell signaling. These studies provide a novel regulatory mechanism for phospholipid-induced changes in the microfilament structure and cell function and suggest that LPA could be an intracellular regulator of the actin cytoskeleton.     

Suction pressure can induce uncoupling of the plasma membrane from cortical actin

We tested the hypothesis that a pressure difference can cause blebbing associated with uncoupling of the plasma membrane from the cortical actin, a phenomenon found earlier in locomoting blebbing Walker carcinosarcoma cells. Untreated, initially spherical Walker carcinosarcoma cells were exposed to suction pressure by partial aspiration into micropipettes. The suction pressure required to induce blebbing was in the range of 0.9-3 cm H2O, i.e., somewhat lower than the increase in intracellular pressure measured before formation of protrusions in Amoeba proteus (Yanai et al., Cell Motil. Cytoskeleton 33, 22-29, 1996). The response was temperature-dependent, blebbing occurring more frequently at 37 degrees C than at room temperature. Blebbing was associated with formation of cytoplasmic actin layers, restriction rings and/or of gaps in the plasma membrane-associated cortical actin. The results support the view that blebbing associated with uncoupling of cortical actin and plasma membrane as observed in locomoting cells can be caused by a pressure gradient.     

Receptor- and store-operated mechanisms of calcium entry during the nanosecond electric pulse-induced cellular response

Nanosecond electric pulses have been shown to open nanopores in the cell plasma membrane by fluorescent imaging of calcium uptake and fluorescent dyes, including propidium (Pr) iodide and YO-PRO-1 (YP₁). Recently, we demonstrated that nsEPs also induce the phosphoinositide intracellular signaling cascade by phosphatidylinositol-4,5-bisphosphate (PIP₂) depletion resulting in physiological responses similar to those observed following stimulation of G_q11-coupled receptors. In this paper, we explore the role of receptor- and store-operated calcium entry (ROCE/SOCE) mechanisms in the observed response of cells to nsEP. We show that addition of the ROCE/SOCE and transient receptor potential channel (TRPC) blocker gadolinium (Gd³⁺, 300 μM) slows PIP2 depletion following 1 and 20 nsEP exposures. Lipid rafts, regions of the plasma membrane rich in PIP₂ and TRPC, are also disrupted by nsEP exposure suggesting that ROCE/SOCE mechanisms are likely impacted. Reducing the expression of stromal interaction molecule 1 (STIM1) protein, a key protein in ROCE and SOCE, in cells exposure to nsEP resulted in a reduction in induced intracellular calcium rise. Additionally, after exposure to 1 and 20 nsEPs (16.2 kV/cm, 5 Hz), intracellular calcium rises were significantly reduced by the addition of GD³⁺ and SKF-96365 (1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy] ethyl-1H-imidazole hydrochloride, 100 μM), a blocker of STIM1 interaction. However, using similar nsEP exposure parameters, SKF-96365 was less effective at reducing YP₁ uptake compared to Gd³⁺. Thus, it is possible that SKF-96365 could block STIM1 interactions within the cell, while Gd³⁺ could acts on TRPC/nanopores from outside of the cell. Our results present evidence of nsEP induces ROCE and SOCE mechanisms and demonstrate that YP₁ and Ca²⁺ cannot be used solely as markers of nsEP-induced nanoporation.     

Functional organization of TRPC-Ca channels and regulation of calcium microdomains

TRP family of proteins are components of unique cation channels that are activated in response to diverse stimuli ranging from growth factor and neurotransmitter stimulation of plasma membrane receptors to a variety of chemical and sensory signals. This review will focus on members of the TRPC sub-family (TRPC1–TRPC7) which currently appear to be the strongest candidates for the enigmatic Ca²⁺ influx channels that are activated in response to stimulation of plasma membrane receptors which result in phosphatidyl inositol-(4,5)-bisphosphate (PIP₂) hydrolysis, generation of IP₃ and DAG, and IP₃-induced Ca²⁺ release from the intracellular Ca²⁺ store via inositol trisphosphate receptor (IP₃R). Homomeric or selective heteromeric interactions between TRPC monomers generate distinct channels that contribute to store-operated as well as store-independent Ca²⁺ entry mechanisms. The former is regulated by the emptying/refilling of internal Ca²⁺ store(s) while the latter depends on PIP₂ hydrolysis (due to changes in PIP₂ per se or an increase in diacylglycerol, DAG). Although the exact physiological function of TRPC channels and how they are regulated has not yet been conclusively established, it is clear that a variety of cellular functions are controlled by Ca²⁺ entry via these channels. Thus, it is critical to understand how cells coordinate the regulation of diverse TRPC channels to elicit specific physiological functions. It is now well established that segregation of TRPC channels mediated by interactions with signaling and scaffolding proteins, determines their localization and regulation in functionally distinct cellular domains. Furthermore, both protein and lipid components of intracellular and plasma membranes contribute to the organization of these microdomains. Such organization serves as a platform for the generation of spatially and temporally dictated [Ca²⁺]_i signals which are critical for precise control of downstream cellular functions.     

Role of Phosphatidylinositol 4,5-Bisphosphate in Ras/Rac-Induced Disruption of the Cortactin-Actomyosin II Complex and Malignant Transformation

Oncogenic Ras mutants such as v-Ha-Ras cause a rapid rearrangement of actin cytoskeleton during malignant transformation of fibroblasts or epithelial cells. Both PI-3 kinase and Rac are required for Ras-induced malignant transformation and membrane ruffling. However, the signal transduction pathway(s) downstream of Rac that leads to membrane ruffling and other cytoskeletal change(s) as well as the exact biochemical nature of the cytoskeletal change remain unknown. Cortactin/EMS1 is the first identified molecule that is dissociated in a Rac–phosphatidylinositol 4,5-biphosphate (PIP₂)-dependent manner from the actin-myosin II complex during Ras-induced malignant transformation; either the PIP₂ binder HS1 or the Rac blocker SCH51344 restores the ability of EMS1 to bind the complex and suppresses the oncogenicity of Ras. Furthermore, while PIP₂ inhibits the actin-EMS1 interaction, HS1 reverses the PIP₂ effect. Thus, we propose that PIP₂, an end-product of the oncogenic Ras/PI-3 kinase/Rac pathway, serves as a second messenger in the Ras/Rac-induced disruption of the actin cytoskeleton and discuss the anticancer drug potential of PIP₂-binding molecules.     

Overexpression of PPK-1, the Caenorhabditis elegans Type I PIP kinase, inhibits growth cone collapse in the developing nervous system and causes axonal degeneration in adults

Growth cones are dynamic membrane structures that migrate to target tissue by rearranging their cytoskeleton in response to environmental cues. The lipid phosphatidylinositol (4,5) bisphosphate (PIP₂) resides on the plasma membrane of all eukaryotic cells and is thought to be required for actin cytoskeleton rearrangements. Thus PIP₂ is likely to play a role during neuron development, but this has never been tested in vivo. In this study, we have characterized the PIP₂ synthesizing enzyme Type I PIP kinase (ppk-1) in Caenorhabditis elegans. PPK-1 is strongly expressed in the nervous system, and can localize to the plasma membrane. We show that PPK-1 purified from C. elegans can generate PIP₂in vitro and that overexpression of the kinase causes an increase in PIP₂ levels in vivo. In developing neurons, PPK-1 overexpression leads to growth cones that become stalled, produce ectopic membrane projections, and branched axons. Once neurons are established, PPK-1 overexpression results in progressive membrane overgrowth and degeneration during adulthood. These data suggest that overexpression of the Type I PIP kinase inhibits growth cone collapse, and that regulation of PIP₂ levels in established neurons may be important to maintain structural integrity and prevent neuronal degeneration.