Spatial control of actin-based motility through plasmalemmal PtdIns(4,5)P2-rich raft assemblies

The interactions of cells with their environment involve regulated actin-based motility at defined positions along the cell surface. Sphingolipid- and cholesterol-dependent microdomains (rafts) order proteins at biological membranes, and have been implicated in most signalling processes at the cell surface. Many membrane-bound components that regulate actin cytoskeleton dynamics and cell-surface motility associate with PtdIns(4,5)P(2)-rich lipid rafts. Although raft integrity is not required for substrate-directed cell spreading, or to initiate signalling for motility, it is a prerequisite for sustained and organized motility. Plasmalemmal rafts redistribute rapidly in response to signals, triggering motility. This process involves the removal of rafts from sites that are not interacting with the substrate, apparently through endocytosis, and a local accumulation at sites of integrin-mediated substrate interactions. PtdIns(4,5)P(2)-rich lipid rafts can assemble into patches in a process depending on PtdIns(4,5)P(2), Cdc42 (cell-division control 42), N-WASP (neural Wiskott-Aldrich syndrome protein) and actin cytoskeleton dynamics. The raft patches are sites of signal-induced actin assembly, and their accumulation locally promotes sustained motility. The patches capture microtubules, which promote patch clustering through PKA (protein kinase A), to steer motility. Raft accumulation at the cell surface, and its coupling to motility are influenced greatly by the expression of intrinsic raft-associated components that associate with the cytosolic leaflet of lipid rafts. Among them, GAP43 (growth-associated protein 43)-like proteins interact with PtdIns(4,5)P(2) in a Ca(2+)/calmodulin and PKC (protein kinase C)-regulated manner, and function as intrinsic determinants of motility and anatomical plasticity. Plasmalemmal PtdIns(4,5)P(2)-rich raft assemblies thus provide powerful organizational principles for tight spatial and temporal control of signalling in motility.     

New EMBO members' review: actin cytoskeleton regulation through modulation of PI(4,5)P(2) rafts

The phosphoinositide lipid PI(4,5)P(2) is now established as a key cofactor in signaling to the actin cytoskeleton and in vesicle trafficking. PI(4,5)P(2) accumulates at membrane rafts and promotes local co-recruitment and activation of specific signaling components at the cell membrane. PI(4,5)P(2) rafts may thus be platforms for local regulation of morphogenetic activity at the cell membrane. Raft PI(4,5)P(2) is regulated by lipid kinases (PI5-kinases) and lipid phosphatases (e.g. synaptojanin). In addition, GAP43-like proteins have recently emerged as a group of PI(4,5)P(2) raft-modulating proteins. These locally abundant proteins accumulate at inner leaflet plasmalemmal rafts where they bind to and co-distribute with PI(4,5)P(2), and promote actin cytoskeleton accumulation and dynamics. In keeping with their proposed role as positive modulators of PI(4,5)P(2) raft function, GAP43-like proteins confer competence for regulated morphogenetic activity on cells that express them. Their function has been investigated extensively in the nervous system, where their expression promotes neurite outgrowth, anatomical plasticity and nerve regeneration. Extrinsic signals and intrinsic factors may thus converge to modulate PI(4,5)P(2) rafts, upstream of regulated activity at the cell surface.     

GAP43, MARCKS, and CAP23 modulate PI(4,5)P(2) at plasmalemmal rafts, and regulate cell cortex actin dynamics through a common mechanism

The dynamic properties of the cell cortex and its actin cytoskeleton determine important aspects of cell behavior and are a major target of cell regulation. GAP43, myristoylated alanine-rich C kinase substrate (MARCKS), and CAP23 (GMC) are locally abundant, plasmalemma-associated PKC substrates that affect actin cytoskeleton. Their expression correlates with morphogenic processes and cell motility, but their role in cortex regulation has been difficult to define mechanistically. We now show that the three proteins accumulate at rafts, where they codistribute with PI(4,5)P(2), and promote its retention and clustering. Binding and modulation of PI(4, 5)P(2) depended on the basic effector domain (ED) of these proteins, and constructs lacking the ED functioned as dominant inhibitors of plasmalemmal PI(4,5)P(2) modulation. In the neuron-like cell line, PC12, NGF- and substrate-induced peripheral actin structures, and neurite outgrowth were greatly augmented by any of the three proteins, and suppressed by DeltaED mutants. Agents that globally mask PI(4,5)P(2) mimicked the effects of GMC on peripheral actin recruitment and cell spreading, but interfered with polarization and process formation. Dominant negative GAP43(DeltaED) also interfered with peripheral nerve regeneration, stimulus-induced nerve sprouting and control of anatomical plasticity at the neuromuscular junction of transgenic mice. These results suggest that GMC are functionally and mechanistically related PI(4,5)P(2) modulating proteins, upstream of actin and cell cortex dynamics regulation.     

Regulation of podosomes by integrin alphavbeta3 and Rho GTPase-facilitated phosphoinositide signaling

In osteoclasts, polyphosphoinositides such as phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5 trisphosphate (PI(3,4,5)P3) are produced in response to integrin alphavbeta3 signaling and they have a critical role in actin cytoskeleton remodeling. The levels of PI(4,5)P2 and PI(3,4,5)P3 are regulated by Rho GTPase through the activation of phosphatidylinositol 4-phosphate 5-kinase (PI4P-5 kinase) and phospatidylinositol 3-kinase (PI3 kinase), respectively. Interaction of PI(4,5)P2 with gelsolin and Wiscott-Aldrich syndrome protein (WASP) is critical for podosome assembly/disassembly and actin ring formation in osteoclasts. Interaction of PI(3,4,5)P3 with gelsolin functions in orchestrating the podosome signaling complex consisting of several key signaling molecules. Gelsolin deficiency has been shown to block podosome assembly and motility in mouse osteoclasts. However, these osteoclasts are able to form a WASP-containing actin ring and retain their resorptive function. The TAT-mediated delivery of gelsolin phosphoinositide-binding domains into osteoclasts resulted in production of podosome clusters and disruption of actin ring formation. Hence, these osteoclasts were hypomotile and less resorptive. Our observations suggest that both PI(4,5)P2 and PI(3,4,5)P3 are involved in regulating osteoclast functions through modulation of severing, capping, and nucleating functions of actin-binding proteins.     

Regulation of gating and rundown of HCN hyperpolarization-activated channels by exogenous and endogenous PIP2

The voltage dependence of activation of the HCN hyperpolarization-activated cation channels is shifted in inside-out patches by -40 to -60 mV relative to activation in intact cells, a phenomenon referred to as rundown. Less than 20 mV of this hyperpolarizing shift can be due to the influence of the canonical modulator of HCN channels, cAMP. Here we study the role of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) in HCN channel rundown, as hydrolysis of PI(4,5)P(2) by lipid phosphatases is thought to underlie rundown of several other channels. We find that bath application of exogenous PI(4,5)P(2) reverses the effect of rundown, producing a large depolarizing shift in HCN2 activation. A synthetic short chain analogue of PI(4,5)P(2), dioctanoyl phosphatidylinositol 4,5-bisphosphate, shifts the HCN2 activation curve to more positive potentials in a dose-dependent manner. Other dioctanoyl phosphatidylinositides with one or more phosphates on the lipid headgroup also shift activation, although phosphatidylinositol (PI) is ineffective. Several lines of evidence suggest that HCN2 is also regulated by endogenous PI(4,5)P(2): (a) blockade of phosphatases slows the hyperpolarizing shift upon patch excision; (b) application of an antibody that binds and depletes membrane PIP(2) causes a further hyperpolarizing shift in activation; (c) the shift in activation upon patch excision can be partially reversed by MgATP; and (d) the effect of MgATP is blocked by wortmannin, an inhibitor of PI kinases. Finally, recordings from rabbit sinoatrial cells demonstrate that diC(8) PI(4,5)P(2) delays the rundown of native HCN currents. Thus, both native and recombinant HCN channels are regulated by PI(4,5)P(2).     

Hydrolysis of phosphatidylinositol 4,5-bisphosphate mediates calcium-induced inactivation of TRPV6 channels

TRPV6 is a member of the transient receptor potential superfamily of ion channels that facilitates Ca(2+) absorption in the intestines. These channels display high selectivity for Ca(2+), but in the absence of divalent cations they also conduct monovalent ions. TRPV6 channels have been shown to be inactivated by increased cytoplasmic Ca(2+) concentrations. Here we studied the mechanism of this Ca(2+)-induced inactivation. Monovalent currents through TRPV6 substantially decreased after a 40-s application of Ca(2+), but not Ba(2+). We also show that Ca(2+), but not Ba(2+), influx via TRPV6 induces depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2) or PIP(2)) and the formation of inositol 1,4,5-trisphosphate. Dialysis of DiC(8) PI(4,5)P(2) through the patch pipette inhibited Ca(2+)-dependent inactivation of TRPV6 currents in whole-cell patch clamp experiments. PI(4,5)P(2) also activated TRPV6 currents in excised patches. PI(4)P, the precursor of PI(4,5)P(2), neither activated TRPV6 in excised patches nor had any effect on Ca(2+)-induced inactivation in whole-cell experiments. Conversion of PI(4,5)P(2) to PI(4)P by a rapamycin-inducible PI(4,5)P(2) 5-phosphatase inhibited TRPV6 currents in whole-cell experiments. Inhibiting phosphatidylinositol 4 kinases with wortmannin decreased TRPV6 currents and Ca(2+) entry into TRPV6-expressing cells. We propose that Ca(2+) influx through TRPV6 activates phospholipase C and the resulting depletion of PI(4,5)P(2) contributes to the inactivation of TRPV6.     

Ca-dependent nonsecretory vesicle fusion in a secretory cell

We have compared Ca-dependent exocytosis in excised giant membrane patches and in whole-cell patch clamp with emphasis on the rat secretory cell line, RBL. Stable patches of 2-4 pF are easily excised from RBL cells after partially disrupting actin cytoskeleton with latrunculin A. Membrane fusion is triggered by switching the patch to a cytoplasmic solution containing 100-200 microM free Ca. Capacitance and amperometric recording show that large secretory granules (SGs) containing serotonin are mostly lost from patches. Small vesicles that are retained (non-SGs) do not release serotonin or other substances detected by amperometry, although their fusion is reduced by tetanus toxin light chain. Non-SG fusion is unaffected by N-ethylmaleimide, phosphatidylinositol-4,5-bis-phosphate (PI(4,5)P(2)) ligands, such as neomycin, a PI-transfer protein that can remove PI from membranes, the PI(3)-kinase inhibitor LY294002 and PI(4,5)P(2), PI(3)P, and PI(4)P antibodies. In patch recordings, but not whole-cell recordings, fusion can be strongly reduced by ATP removal and by the nonspecific PI-kinase inhibitors wortmannin and adenosine. In whole-cell recording, non-SG fusion is strongly reduced by osmotically induced cell swelling, and subsequent recovery after shrinkage is then inhibited by wortmannin. Thus, membrane stretch that occurs during patch formation may be a major cause of differences between excised patch and whole-cell fusion responses. Regarding Ca sensors for non-SG fusion, fusion remains robust in synaptotagmin (Syt) VII-/- mouse embryonic fibroblasts (MEFs), as well as in PLCdelta1, PLC delta1/delta4, and PLCgamma1-/- MEFs. Thus, Syt VII and several PLCs are not required. Furthermore, the Ca dependence of non-SG fusion reflects a lower Ca affinity (K(D) approximately 71 microM) than expected for these C2 domain-containing proteins. In summary, we find that non-SG membrane fusion behaves and is regulated substantially differently from SG fusion, and we have identified an ATP-dependent process that restores non-SG fusion capability after it is perturbed by membrane stretch or cell dilation.     

The LIM protein Ajuba regulates phosphatidylinositol 4,5-bisphosphate levels in migrating cells through an interaction with and activation of PIPKI alpha

The phosphoinositide phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] regulates the activity of many actin-binding proteins and as such is an important modulator of cytoskeleton organization during cell migration, for example. In migrating cells actin remodeling is tightly regulated and localized; therefore, how the PI(4,5)P2 level is spatially and temporally regulated is crucial to understanding how it controls cell migration. Here we show that the LIM protein Ajuba contributes to the cellular regulation of PI(4,5)P2 levels by interacting with and activating the enzymatic activity of the PI(4)P 5-kinase (PIPKIalpha), the predominant enzyme in the synthesis of PI(4,5)P2, in a migration stimulus-regulated manner. In migrating primary mouse embryonic fibroblasts (MEFs) from Ajuba(-/-) mice the level of PI(4,5)P2 was decreased with a corresponding increase in the level of the substrate PI(4)P. Reintroduction of Ajuba into these cells normalized PI(4,5)P2 levels. Localization of PI(4,5)P2 synthesis and PIPKIalpha in the leading lamellipodia and membrane ruffles, respectively, of migrating Ajuba(-/-) MEFs was impaired. In vitro, Ajuba dramatically activated the enzymatic activity of PIPKIalpha while inhibiting the activity of PIPKIIbeta. Thus, in addition to its effects upon Rac activity Ajuba can also influence cell migration through regulation of PI(4,5)P2 synthesis through direct activation of PIPKIalpha enzyme activity.     

Phosphatidylinositol 4,5-bisphosphate rescues TRPM4 channels from desensitization

TRPM4 is a Ca(2+)-activated nonselective cation channel that regulates membrane potential in response to intracellular Ca(2+) signaling. In lymphocytes it plays an essential role in shaping the pattern of intracellular Ca(2+) oscillations that lead to cytokine secretion. To better understand its role in this and other physiological processes, we investigated mechanisms by which TRPM4 is regulated. TRPM4 was expressed in ChoK1 cells, and currents were measured in excised patches. Under these conditions, TRPM4 currents were activated by micromolar concentrations of cytoplasmic Ca(2+) and progressively desensitized. Here we show that desensitization can be explained by a loss of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) from the channels. Poly-l-lysine, a PI(4,5)P(2) scavenger, caused rapid desensitization, whereas MgATP, at concentrations that activate lipid kinases, promoted recovery of TRPM4 currents. Application of exogenous PI(4,5)P(2) to the intracellular surface of the patch restored the properties of TRPM4 currents. Our results suggest that PI(4,5)P(2) acts to uncouple channel opening from changes in the transmembrane potential, allowing current activation at physiological voltages. These data argue that hydrolysis of PI(4,5)P(2) underlies desensitization of TRPM4 and support the idea that PI(4,5)P(2) is a general regulator for the gating of TRPM ion channels.     

An association between type Iγ PI4P 5-kinase and Exo70 directs E-cadherin clustering and epithelial polarization

E-Cadherin-mediated formation of adherens junctions (AJs) is essential for the morphogenesis of epithelial cells. However, the mechanisms underlying E-cadherin clustering and AJ maturation are not fully understood. Here we report that type Iγ phosphatidylinositol-4-phosphate 5-kinase (PIPKIγ) associates with the exocyst via a direct interaction with Exo70, the exocyst subunit that guides the polarized targeting of exocyst to the plasma membrane. By means of this interaction, PIPKIγ mediates the association between E-cadherin and Exo70 and determines the targeting of Exo70 to AJs. Further investigation revealed that Exo70 is necessary for clustering of E-cadherin on the plasma membrane and extension of nascent E-cadherin adhesions, which are critical for the maturation of cohesive AJs. In addition, we observed phosphatidylinositol-4,5-bisphosphate (PI4,5P(2)) accumulation at E-cadherin clusters during the assembly of E-cadherin adhesions. PIPKIγ-generated PI4,5P(2) is required for recruiting Exo70 to newly formed E-cadherin junctions and facilitates the assembly and maturation of AJs. These results support a model in which PIPKIγ and PIPKIγ-generated PI4,5P(2) pools at nascent E-cadherin contacts cue Exo70 targeting and orient the tethering of exocyst-associated E-cadherin. This could be an important mechanism that regulates E-cadherin clustering and AJ maturation, which is essential for the establishment of solid, polarized epithelial structures.     

A CAAX motif can compensate for the PH domain of Num1 for cortical dynein attachment

During mitosis in budding yeast, cortically anchored dynein exerts pulling forces on cytoplasmic microtubules, moving the mitotic spindle into the mother-bud neck. Anchoring of dynein requires the cortical patch protein Num1, which is hypothesized to interact with PI(4,5)P₂ via its C-terminal pleckstrin homology (PH) domain. Here we show that the PH domain and PI(4,5)P₂ are required for the cortical localization of Num1, but are not sufficient to mediate the cortical assembly of Num1 patches. A GFP fusion to the PH domain localizes to the cortex in foci containing ~2 molecules, whereas patches of full-length Num1-GFP contain ~14 molecules. A membrane targeting sequence containing the CAAX motif from the yeast Ras2 protein can compensate for the PH domain to target Num1 to the plasma membrane as discrete patches. The CAAX-targeted Num1 exhibits overlapping but largely distinct localization from wild-type Num1. However, it is fully functional in the dynein pathway. More importantly, cortical PI(4,5)P₂ is dispensable for the localization and function of the CAAX-targeted Num1. Together, these results demonstrate that cortical assembly of Num1 into functional dynein-anchoring patches is independent of its interaction with PI(4,5)P₂.     

A phosphatidylinositol (4,5)-bisphosphate binding site within mu2-adaptin regulates clathrin-mediated endocytosis

The clathrin adaptor complex AP-2 serves to coordinate clathrin-coated pit assembly with the sorting of transmembrane cargo proteins at the plasmalemma. How precisely AP-2 assembly and cargo protein recognition at sites of endocytosis are regulated has remained unclear, but recent evidence implicates phosphoinositides, in particular phosphatidylinositol (4,5)-bisphosphate (PI[4,5]P2), in these processes. Here we have identified and functionally characterized a conserved binding site for PI(4,5)P2 within mu2-adaptin, the medium chain of the clathrin adaptor complex AP-2. Mutant mu2 lacking a cluster of conserved lysine residues fails to bind PI(4,5)P2 and to compete the recruitment of native clathrin/AP-2 to PI(4,5)P2-containing liposomes or to presynaptic membranes. Moreover, we show that expression of mutant mu2 inhibits receptor-mediated endocytosis in living cells. We suggest that PI(4,5)P2 binding to mu2-adaptin regulates clathrin-mediated endocytosis and thereby may contribute to structurally linking cargo recognition to coat formation.     

Initiation of cell locomotility is a morphogenetic checkpoint in thyroid epithelial cells regulated by ERK and PI3-kinase signals.

Epithelial locomotility is a fundamental determinant of tissue patterning that is subject to strict physiological regulation. The current study sought to identify cellular signals that initiate cell migration in cultured thyroid epithelial cells. Porcine thyroid cells cultured as 3-dimensional follicles convert to 2-dimensional monolayers when deprived of agents that stimulate cAMP/PKA signaling. This morphogenetic event is driven by the activation of cell-on-substrate locomotility, providing a convenient assay for events that regulate the initiation of locomotion. In this system, the extracellular signal regulated kinase (ERK) pathway became activated as follicles converted to monolayer, as demonstrated by immunoblotting for activation-specific phosphorylation and nuclear accumulation of ERK. Inhibition of ERK activation using the drug PD98059 effectively prevented cells from beginning to migrate. PD98059 inhibited cell spreading, actin filament reorganization and the assembly of focal adhesions, cellular events that mediate the initiation of thyroid cell locomotility. Akt (PKB) signaling was also activated during follicle-to-monolayer conversion and the phosphoinositide 3-kinase (PI3-kinase) inhibitor, wortmannin, also blocked the initiation of cell movement. Wortmannin did not, however, block activation of ERK signaling. These findings, therefore, identify the ERK and PI3-kinase signaling pathways as important stimulators of thyroid cell locomotility. These findings are incorporated into a model where the initiation of thyroid cell motility constitutes a morphogenetic checkpoint regulated by coordinated changes in stimulatory (ERK, PI3-kinase) and tonic inhibitory (cAMP/PKA) signaling pathways.     

Norepinephrine and endothelin activate diacylglycerol kinases in caveolae/rafts of rat mesenteric arteries: agonist-specific role of PI3-kinase

The phosphatidylinositol (PI) signaling pathway mediates norepinephrine (NE)- and endothelin-1 (ET-1)-stimulated vascular smooth muscle contraction through an inositol-trisphosphate-induced rise in intracellular calcium and diacylglycerol (DG) activation of protein kinase C (PKC). Subsequent activation of DG kinases (DGKs) metabolizes DG to phosphatidic acid (PA), potentially regulating PKC activity. Because precise regulation and spatial restriction of the PI pathway is necessary for specificity, we have investigated whether this occurs within caveolae/rafts, specialized plasma membrane microdomains implicated in vascular smooth muscle contraction. We show that components of the PI signaling cascade-phosphatidylinositol 4,5-bisphosphate (PIP(2)), PA, and DGK-theta are present in caveolae/rafts prepared from rat mesenteric small arteries. Stimulation with NE or ET-1 induced [(33)P]PIP(2) hydrolysis solely within caveolae/rafts. NE stimulated an increase in DGK activity in caveolae/rafts alone, whereas ET-1 activated DGK in caveolae/rafts and noncaveolae/rafts; however, [(33)P]PA increased in all fractions with both agonists. Previously, we reported that NE activated DGK-theta in a phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner; here, we describe PI3-kinase-dependent DGK activation and [(33)P]PA production in caveolae/rafts in response to NE but not ET-1. Additionally, PKB, a potential activator of DGK-theta, translocated to caveolae/rafts in response to NE but not ET-1, and PI3-kinase inhibition prevented this. Furthermore, PI3-kinase inhibition reduced the sensitivity of contraction to NE but not ET-1. Our study shows that caveolae/rafts are major sites of vasoconstrictor hormone activation of the PI pathway in intact small arteries and suggest a link between lipid signaling events within caveolae/rafts and contraction.     

Control of lipid organization and actin assembly during clathrin-mediated endocytosis by the cytoplasmic tail of the rhomboid protein Rbd2

Clathrin-mediated endocytosis (CME) is facilitated by a precisely regulated burst of actin assembly. PtdIns(4,5)P2 is an important signaling lipid with conserved roles in CME and actin assembly regulation. Rhomboid family multipass transmembrane proteins regulate diverse cellular processes; however, rhomboid-mediated CME regulation has not been described. We report that yeast lacking the rhomboid protein Rbd2 exhibit accelerated endocytic-site dynamics and premature actin assembly during CME through a PtdIns(4,5)P2-dependent mechanism. Combined genetic and biochemical studies showed that the cytoplasmic tail of Rbd2 binds directly to PtdIns(4,5)P2 and is sufficient for Rbd2''s role in actin regulation. Analysis of an Rbd2 mutant with diminished PtdIns(4,5)P2-binding capacity indicates that this interaction is necessary for the temporal regulation of actin assembly during CME. The cytoplasmic tail of Rbd2 appears to modulate PtdIns(4,5)P2 distribution on the cell cortex. The syndapin-like F-BAR protein Bzz1 functions in a pathway with Rbd2 to control the timing of type 1 myosin recruitment and actin polymerization onset during CME. This work reveals that the previously unstudied rhomboid protein Rbd2 functions in vivo at the nexus of three highly conserved processes: lipid regulation, endocytic regulation, and cytoskeletal function.     

Phosphoinositides suppress gamma-secretase in both the detergent-soluble and -insoluble states

gamma-Secretase is an aspartic protease that hydrolyzes type I membrane proteins within the hydrophobic environment of the lipid bilayer. Using the CHAPSO-solubilized gamma-secretase assay system, we previously found that gamma-secretase activity was sensitive to the concentrations of detergent and phosphatidylcholine. This strongly suggests that the composition of the lipid bilayer has a significant impact on the activity of gamma-secretase. Recently, level of secreted beta-amyloid protein was reported to be attenuated by increasing levels of phosphatidylinositol 4,5-diphosphate (PI(4,5)P2) in cultured cells. However, it is not clear whether PI(4,5)P2 has a direct effect on gamma-secretase activity. In this study, we found that phosphoinositides directly inhibited CHAPSO-solubilized gamma-secretase activity. Interestingly, neither phosphatidylinositol nor inositol triphosphate altered gamma-secretase activity. PI(4,5)P2 was also found to inhibit gamma-secretase activity in CHAPSO-insoluble membrane microdomains (rafts). Kinetic analysis of beta-amyloid protein production in the presence of PI(4,5)P2 suggested a competitive inhibition. Even though phosphoinositides are minor phospholipids of the membrane, the concentration of PI(4,5)P2 within the intact membrane has been reported to be in the range of 4-8 mm. The presence of PI(4,5)P2-rich rafts in the membrane has been reported in a range of cell types. Furthermore, gamma-secretase is enriched in rafts. Taking these data together, we propose that phosphoinositides potentially regulate gamma-secretase activity by suppressing its association with the substrate.     

Elimination of host cell PtdIns(4,5)P(2) by bacterial SigD promotes membrane fission during invasion by Salmonella

Salmonella invades mammalian cells by inducing membrane ruffling and macropinocytosis through actin remodelling. Because phosphoinositides are central to actin assembly, we have studied the dynamics of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)) in HeLa cells during invasion by Salmonella typhimurium. Here we show that the outermost parts of the ruffles induced by invasion show a modest enrichment in PtdIns(4,5)P(2), but that PtdIns(4,5)P(2) is virtually absent from the invaginating regions. Rapid disappearance of PtdIns(4,5)P(2) requires the expression of the Salmonella phosphatase SigD (also known as SopB). Deletion of SigD markedly delays fission of the invaginating membranes, indicating that elimination of PtdIns(4,5)P(2) may be required for rapid formation of Salmonella-containing vacuoles. Heterologous expression of SigD is sufficient to promote the disappearance of PtdIns(4,5)P(2), to reduce the rigidity of the membrane skeleton, and to induce plasmalemmal invagination and fission. Hydrolysis of PtdIns(4,5)P(2) may be a common and essential feature of membrane fission during several internalization processes including invasion, phagocytosis and possibly endocytosis.     

A novel GRK2/HDAC6 interaction modulates cell spreading and motility

Cell motility and adhesion involves dynamic microtubule (MT) acetylation/deacetylation, a process regulated by enzymes as HDAC6, a major cytoplasmic α-tubulin deacetylase. We identify G protein-coupled receptor kinase 2 (GRK2) as a key novel stimulator of HDAC6. GRK2, which levels inversely correlate with the extent of α-tubulin acetylation in epithelial cells and fibroblasts, directly associates with and phosphorylates HDAC6 to stimulate α-tubulin deacetylase activity. Remarkably, phosphorylation of GRK2 itself at S670 specifically potentiates its ability to regulate HDAC6. GRK2 and HDAC6 colocalize in the lamellipodia of migrating cells, leading to local tubulin deacetylation and enhanced motility. Consistently, cells expressing GRK2-K220R or GRK2-S670A mutants, unable to phosphorylate HDAC6, exhibit highly acetylated cortical MTs and display impaired migration and protrusive activity. Finally, we find that a balanced, GRK2/HDAC6-mediated regulation of tubulin acetylation differentially modulates the early and late stages of cellular spreading. This novel GRK2/HDAC6 functional interaction may have important implications in pathological contexts.     

Imaging metabolism of phosphatidylinositol 4,5-bisphosphate in T-cell GM1-enriched domains containing Ras proteins

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and Ras proteins are involved in signalling pathways originating at the plasma membrane. The localisation and metabolism of PI(4,5)P(2) was studied in Jurkat T cells using fluorescence microscopic imaging with EGFP-tagged and antibody probes. Software was developed to objectively quantitate colocalisation and was used to show that plasma membrane PI(4,5)P(2) was enriched in lipid raft-containing patches of GM1 ganglioside, formed by crosslinking cholera toxin B-subunit (CT-B). The PI(4,5)P(2) metabolites phosphatidylinositol 3,4,5-trisphosphate and diacylglycerol appeared in plasma membrane CT-B-GM1 patches upon induction of signalling. Transferrin receptor and the CD45 tyrosine phosphatase did not colocalise with CT-B-GM1 patches, whereas the tyrosine kinase Lck, the scaffolding protein LAT, and endogenous Ras proteins did partially colocalise with CT-B-GM1 patches as did transfected EGFP-K-Ras(4B) and EGFP-H-Ras. The results demonstrate that T-cell PI(4,5)P(2) metabolism is occurring in GM1-enriched domains and that Ras proteins are present in these domains in vivo.     

Phosphatidylinositol 4,5-bisphosphate,cholesterol, and fatty acids modulate the calcium-activated chloride channel TMEM16A (ANO1)

The TMEM16A-mediated Ca²⁺-activated Cl^? current drives several important physiological functions. Membrane lipids regulate ion channels and transporters but their influence on members of the TMEM16 family is poorly understood. Here we have studied the regulation of TMEM16A by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), cholesterol, and fatty acids using patch clamp, biochemistry and fluorescence microscopy. We found that depletion of membrane PI(4,5)P2 causes a decline in TMEM16A current that is independent of cytoskeleton, but is partially prevented by removing intracellular Ca²⁺. On the other hand, supplying PI(4,5)P2 to inside-out patches attenuated channel rundown and/or partially rescued activity after channel rundown. Also, depletion (with methyl-β-cyclodextrin M-βCD) or restoration (with M-βCD + cholesterol) of membrane cholesterol slows down the current decay observed after reduction of PI(4,5)P2. Neither depletion nor restoration of cholesterol change PI(4,5)P2 content. However, M-βCD alone transiently increases TMEM16A activity and dampens rundown whereas M-βCD + cholesterol increases channel rundown. Thus, PI(4,5)P2 is required for TMEM16A function while cholesterol directly and indirectly via a PI(4,5)P2-independent mechanism regulate channel function. Stearic, arachidonic, oleic, docosahexaenoic, and eicosapentaenoic fatty acids as well as methyl stearate inhibit TMEM16A in a dose- and voltage-dependent manner. Phosphatidylserine, a phospholipid whose hydrocarbon tails contain stearic and oleic acids also inhibits TMEM16A. Finally, we show that TMEM16A remains in the plasma membrane after treatment with M-βCD, M-βCD + cholesterol, oleic, or docosahexaenoic acids. Thus, we propose that lipids and fatty acids regulate TMEM16A channels through a membrane-delimited protein-lipid interaction.