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Unbalancing the phosphatidylinositol-4,5-bisphosphate-cofilin interaction impairs cell steering.

ABSTRACT: Cofilin is a key player in actin dynamics during cell migration. Its activity is regulated by (de)phosphorylation, pH, and binding to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P(2)]. Here, we here use a human cofilin-1 (D122K) mutant with increased binding affinity for PI(4,5)P(2) and slower release from the plasma membrane to study the role of the PI(4,5)P(2)-cofilin interaction in migrating cells. In fibroblasts in a background of endogenous cofilin, D122K cofilin expression negatively affects cell turning frequency. In carcinoma cells with down-regulated endogenous cofilin, D122K cofilin neither rescues the drastic morphological defects nor restores the effects in cell turning capacity, unlike what has been reported for wild-type cofilin. In cofilin knockdown cells, D122K cofilin expression promotes outgrowth of an existing lamellipod in response to epidermal growth factor (EGF) but does not result in initiation of new lamellipodia. This indicates that, next to phospho- and pH regulation, the normal release kinetics of cofilin from PI(4,5)P(2) is crucial as a local activation switch for lamellipodia initiation and as a signal for migrating cells to change direction in response to external stimuli. Our results demonstrate that the PI(4,5)P(2) regulatory mechanism, that is governed by EGF-dependent phospholipase C activation, is a determinant for the spatial and temporal control of cofilin activation required for lamellipodia initiation.

SUBMITTER: Leyman S

PROVIDER: S-EPMC2770939 | biostudies-literature |

REPOSITORIES: biostudies-literature

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A reducing milieu renders cofilin insensitive to phosphatidylinositol 4,5-bisphosphate (PIP2) inhibition.

Project description:Oxidative stress can lead to T cell hyporesponsiveness. A reducing micromilieu (e.g. provided by dendritic cells) can rescue T cells from such oxidant-induced dysfunction. However, the reducing effects on proteins leading to restored T cell activation remained unknown. One key molecule of T cell activation is the actin-remodeling protein cofilin, which is dephosphorylated on serine 3 upon T cell costimulation and has an essential role in formation of mature immune synapses between T cells and antigen-presenting cells. Cofilin is spatiotemporally regulated; at the plasma membrane, it can be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2). Here, we show by NMR spectroscopy that a reducing milieu led to structural changes in the cofilin molecule predominantly located on the protein surface. They overlapped with the PIP2- but not actin-binding sites. Accordingly, reduction of cofilin had no effect on F-actin binding and depolymerization and did not influence the cofilin phosphorylation state. However, it did prevent inhibition of cofilin activity through PIP2. Therefore, a reducing milieu may generate an additional pool of active cofilin at the plasma membrane. Consistently, in-flow microscopy revealed increased actin dynamics in the immune synapse of untransformed human T cells under reducing conditions. Altogether, we introduce a novel mechanism of redox regulation: reduction of the actin-remodeling protein cofilin renders it insensitive to PIP2 inhibition, resulting in enhanced actin dynamics.

| S-EPMC3795243 | biostudies-literature

Novel Kv7.1-phosphatidylinositol 4,5-bisphosphate interaction sites uncovered by charge neutralization scanning.

Project description:Kv7.1 to Kv7.5 ?-subunits belong to the family of voltage-gated potassium channels (Kv). Assembled with the ?-subunit KCNE1, Kv7.1 conducts the slowly activating potassium current IKs, which is one of the major currents underlying repolarization of the cardiac action potential. A known regulator of Kv7 channels is the lipid phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 increases the macroscopic current amplitude by stabilizing the open conformation of 7.1/KCNE1 channels. However, knowledge about the exact nature of the interaction is incomplete. The aim of this study was the identification of the amino acids responsible for the interaction between Kv7.1 and PIP2. We generated 13 charge neutralizing point mutations at the intracellular membrane border and characterized them electrophysiologically in complex with KCNE1 under the influence of diC8-PIP2. Electrophysiological analysis of corresponding long QT syndrome mutants suggested impaired PIP2 regulation as the cause for channel dysfunction. To clarify the underlying structural mechanism of PIP2 binding, molecular dynamics simulations of Kv7.1/KCNE1 complexes containing two PIP2 molecules in each subunit at specific sites were performed. Here, we identified a subset of nine residues participating in the interaction of PIP2 and Kv7.1/KCNE1. These residues may form at least two binding pockets per subunit, leading to the stabilization of channel conformations upon PIP2 binding.

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Phosphatidylinositol 4,5-bisphosphate optical uncaging potentiates exocytosis.

Project description:Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is essential for exocytosis. Classical ways of manipulating PI(4,5)P2 levels are slower than its metabolism, making it difficult to distinguish effects of PI(4,5)P2 from those of its metabolites. We developed a membrane-permeant, photoactivatable PI(4,5)P2, which is loaded into cells in an inactive form and activated by light, allowing sub-second increases in PI(4,5)P2 levels. By combining this compound with electrophysiological measurements in mouse adrenal chromaffin cells, we show that PI(4,5)P2 uncaging potentiates exocytosis and identify synaptotagmin-1 (the Ca2+ sensor for exocytosis) and Munc13-2 (a vesicle priming protein) as the relevant effector proteins. PI(4,5)P2 activation of exocytosis did not depend on the PI(4,5)P2-binding CAPS-proteins, suggesting that PI(4,5)P2 uncaging may bypass CAPS-function. Finally, PI(4,5)P2 uncaging triggered the rapid fusion of a subset of readily-releasable vesicles, revealing a rapid role of PI(4,5)P2 in fusion triggering. Thus, optical uncaging of signaling lipids can uncover their rapid effects on cellular processes and identify lipid effectors.

| S-EPMC5711374 | biostudies-literature

Profilin interaction with phosphatidylinositol (4,5)-bisphosphate destabilizes the membrane of giant unilamellar vesicles.

Project description:Profilin, a small cytoskeletal protein, and phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] have been implicated in cellular events that alter the cell morphology, such as endocytosis, cell motility, and formation of the cleavage furrow during cytokinesis. Profilin has been shown to interact with PI(4,5)P2, but the role of this interaction is still poorly understood. Using giant unilamellar vesicles (GUVs) as a simple model of the cell membrane, we investigated the interaction between profilin and PI(4,5)P2. A number and brightness analysis demonstrated that in the absence of profilin, molar ratios of PI(4,5)P2 above 4% result in lipid demixing and cluster formations. Furthermore, adding profilin to GUVs made with 1% PI(4,5)P2 leads to the formation of clusters of both profilin and PI(4,5)P2. However, due to the self-quenching of the dipyrrometheneboron difluoride-labeled PI(4,5)P2, we were unable to determine the size of these clusters. Finally, we show that the formation of these clusters results in the destabilization and deformation of the GUV membrane.

| S-EPMC2712054 | biostudies-literature

Reduced phosphatidylinositol 4,5-bisphosphate synthesis impairs inner ear Ca2+ signaling and high-frequency hearing acquisition.

Project description:Phosphatidylinositol phosphate kinase type 1? (PIPKI?) is a key enzyme in the generation of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] and is expressed at high levels in the nervous system. Homozygous knockout mice lacking this enzyme die postnatally within 24 h, whereas PIPKI?(+/-) siblings breed normally and have no reported phenotype. Here we show that adult PIPKI?(+/-) mice have dramatically elevated hearing thresholds for high-frequency sounds. During the first postnatal week we observed a reduction of ATP-dependent Ca(2+) signaling activity in cochlear nonsensory cells. Because Ca(2+) signaling under these conditions depends on inositol-1,4,5-trisphosphate generation from phospholipase C (PLC)-dependent hydrolysis of PI(4,5)P(2), we conclude that (i) PIPKI? is primarily responsible for the synthesis of the receptor-regulated PLC-sensitive PI(4,5)P(2) pool in the cell syncytia that supports auditory hair cells; (ii) spatially graded impairment of this signaling pathway in cochlear nonsensory cells causes a selective alteration in the acquisition of hearing in PIPKI?(+/-) mice. This mouse model also suggests that PIPKI? may determine the level of gap junction contribution to cochlear development.

| S-EPMC3435166 | biostudies-literature

Phosphate position is key in mediating transmembrane ion channel TMEM16A-phosphatidylinositol 4,5-bisphosphate interaction.

Project description:TransMEMbrane 16A (TMEM16A) is a Ca2+-activated Cl- channel that plays critical roles in regulating diverse physiologic processes, including vascular tone, sensory signal transduction, and mucosal secretion. In addition to Ca2+, TMEM16A activation requires the membrane lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). However, the structural determinants mediating this interaction are not clear. Here, we interrogated the parts of the PI(4,5)P2 head group that mediate its interaction with TMEM16A by using patch- and two-electrode voltage-clamp recordings on oocytes from the African clawed frog Xenopus laevis, which endogenously express TMEM16A channels. During continuous application of Ca2+ to excised inside-out patches, we found that TMEM16A-conducted currents decayed shortly after patch excision. Following this rundown, we show that the application of a synthetic PI(4,5)P2 analog produced current recovery. Furthermore, inducible dephosphorylation of PI(4,5)P2 reduces TMEM16A-conducted currents. Application of PIP2 analogs with different phosphate orientations yielded distinct amounts of current recovery, and only lipids that include a phosphate at the 4' position effectively recovered TMEM16A currents. Taken together, these findings improve our understanding of how PI(4,5)P2 binds to and potentiates TMEM16A channels.

| S-EPMC9396059 | biostudies-literature

Phosphatidylinositol 4,5-bisphosphate regulates SNARE-dependent membrane fusion.

Project description:Phosphatidylinositol 4,5-bisphosphate (PI 4,5-P(2)) on the plasma membrane is essential for vesicle exocytosis but its role in membrane fusion has not been determined. Here, we quantify the concentration of PI 4,5-P(2) as approximately 6 mol% in the cytoplasmic leaflet of plasma membrane microdomains at sites of docked vesicles. At this concentration of PI 4,5-P(2) soluble NSF attachment protein receptor (SNARE)-dependent liposome fusion is inhibited. Inhibition by PI 4,5-P(2) likely results from its intrinsic positive curvature-promoting properties that inhibit formation of high negative curvature membrane fusion intermediates. Mutation of juxtamembrane basic residues in the plasma membrane SNARE syntaxin-1 increase inhibition by PI 4,5-P(2), suggesting that syntaxin sequesters PI 4,5-P(2) to alleviate inhibition. To define an essential rather than inhibitory role for PI 4,5-P(2), we test a PI 4,5-P(2)-binding priming factor required for vesicle exocytosis. Ca(2+)-dependent activator protein for secretion promotes increased rates of SNARE-dependent fusion that are PI 4,5-P(2) dependent. These results indicate that PI 4,5-P(2) regulates fusion both as a fusion restraint that syntaxin-1 alleviates and as an essential cofactor that recruits protein priming factors to facilitate SNARE-dependent fusion.

| S-EPMC2483516 | biostudies-literature

Phosphatidylinositol 4,5-bisphosphate and Arf6-regulated membrane traffic.

Project description:ADP-ribosylation factor (Arf) 6 regulates the movement of membrane between the plasma membrane (PM) and a nonclathrin-derived endosomal compartment and activates phosphatidylinositol 4-phosphate 5-kinase (PIP 5-kinase), an enzyme that generates phosphatidylinositol 4,5-bisphosphate (PIP2). Here, we show that PIP2 visualized by expressing a fusion protein of the pleckstrin homology domain from PLCdelta and green fluorescent protein (PH-GFP), colocalized with Arf6 at the PM and on tubular endosomal structures. Activation of Arf6 by expression of its exchange factor EFA6 stimulated protrusion formation, the uptake of PM into macropinosomes enriched in PIP2, and recycling of this membrane back to the PM. By contrast, expression of Arf6 Q67L, a GTP hydrolysis-resistant mutant, induced the formation of PIP2-positive actin-coated vacuoles that were unable to recycle membrane back to the PM. PM proteins, such as beta1-integrin, plakoglobin, and major histocompatibility complex class I, that normally traffic through the Arf6 endosomal compartment became trapped in this vacuolar compartment. Overexpression of human PIP 5-kinase alpha mimicked the effects seen with Arf6 Q67L. These results demonstrate that PIP 5-kinase activity and PIP2 turnover controlled by activation and inactivation of Arf6 is critical for trafficking through the Arf6 PM-endosomal recycling pathway.

| S-EPMC2196179 | biostudies-literature

Phosphatidylinositol 4,5-bisphosphate directs spermatid cell polarity and exocyst localization in Drosophila.

Project description:During spermiogenesis, Drosophila melanogaster spermatids coordinate their elongation in interconnected cysts that become highly polarized, with nuclei localizing to one end and sperm tail growth occurring at the other. Remarkably little is known about the signals that drive spermatid polarity and elongation. Here we identify phosphoinositides as critical regulators of these processes. Reduction of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) by low-level expression of the PIP(2) phosphatase SigD or mutation of the PIP(2) biosynthetic enzyme Skittles (Sktl) results in dramatic defects in spermatid cysts, which become bipolar and fail to fully elongate. Defects in polarity are evident from the earliest stages of elongation, indicating that phosphoinositides are required for establishment of polarity. Sktl and PIP(2) localize to the growing end of the cysts together with the exocyst complex. Strikingly, the exocyst becomes completely delocalized when PIP(2) levels are reduced, and overexpression of Sktl restores exocyst localization and spermatid cyst polarity. Moreover, the exocyst is required for polarity, as partial loss of function of the exocyst subunit Sec8 results in bipolar cysts. Our data are consistent with a mechanism in which localized synthesis of PIP(2) recruits the exocyst to promote targeted membrane delivery and polarization of the elongating cysts.

| S-EPMC2861613 | biostudies-literature

Phosphatidylinositol (4,5) bisphosphate controls T cell activation by regulating T cell rigidity and organization.

Project description:Here we investigate the role of Phosphatidylinositol (4,5) bisphosphate (PIP(2)) in the physiological activation of primary murine T cells by antigen presenting cells (APC) by addressing two principal challenges in PIP(2) biology. First, PIP(2) is a regulator of cytoskeletal dynamics and a substrate for second messenger generation. The relative importance of these two processes needs to be determined. Second, PIP(2) is turned over by multiple biosynthetic and metabolizing enzymes. The joint effect of these enzymes on PIP(2) distributions needs to be determined with resolution in time and space. We found that T cells express four isoforms of the principal PIP(2)-generating enzyme phosphatidylinositol 4-phosphate 5-kinase (PIP5K) with distinct spatial and temporal characteristics. In the context of a larger systems analysis of T cell signaling, these data identify the T cell/APC interface and the T cell distal pole as sites of differential PIP(2) turnover. Overexpression of different PIP5K isoforms, as corroborated by knock down and PIP(2) blockade, yielded an increase in PIP(2) levels combined with isoform-specific changes in the spatiotemporal distributions of accessible PIP(2). It rigidified the T cell, likely by impairing the inactivation of Ezrin Moesin Radixin, delayed and diminished the clustering of the T cell receptor at the cellular interface, reduced the efficiency of T cell proximal signaling and IL-2 secretion. These effects were consistently more severe for distal PIP5K isoforms. Thus spatially constrained cytoskeletal roles of PIP(2) in the control of T cell rigidity and spatiotemporal organization dominate the effects of PIP(2) on T cell activation.

| S-EPMC3214035 | biostudies-literature

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