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Paul A Janmey - One of the best experts on this subject based on the ideXlab platform.
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soft hyaluronic gels promote cell spreading stress fibers focal adhesion and membrane tension by phosphoinositide signaling not traction force
ACS Nano, 2019Co-Authors: Kalpana Mandal, Dikla Razben Aroush, Zachary T Graber, Chan Young Park, Jeffery J Fredberg, Wei Guo, Tobias Baumgart, Paul A JanmeyAbstract:Cells respond to both physical and chemical aspects of their substrate. Whether intracellular signals initiated by physical stimuli are fundamentally different from those elicited by chemical stimuli is an open question. Here, we show that the requirement for a stiff substrate (and, therefore, high cellular tension) for cells to produce large focal adhesions and stress fibers is obviated when a soft substrate contains both hyaluronic acid (HA) and an integrin ligand (collagen I). HA is a major extracellular matrix component that is often up-regulated during wound healing and tumor growth. HA, together with collagen I, promotes hepatocellular carcinoma cell (Huh7) spreading on very soft substrates (300 Pa), resulting in morphology and motility similar to what these cells develop only on stiff substrates (>30 kPa) formed by polyacrylamide that contains collagen but not HA. The effect of HA requires turnover of Polyphosphoinositides and leads to the activation of Akt. The inhibition of Polyphosphoinositide t...
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Soft Hyaluronic Gels Promote Cell Spreading, Stress Fibers, Focal Adhesion, and Membrane Tension by Phosphoinositide Signaling, Not Traction Force
2018Co-Authors: Kalpana Mandal, Dikla Razben Aroush, Zachary T Graber, Chan Young Park, Jeffery J Fredberg, Wei Guo, Tobias Baumgart, Paul A JanmeyAbstract:Cells respond to both physical and chemical aspects of their substrate. Whether intracellular signals initiated by physical stimuli are fundamentally different from those elicited by chemical stimuli is an open question. Here, we show that the requirement for a stiff substrate (and, therefore, high cellular tension) for cells to produce large focal adhesions and stress fibers is obviated when a soft substrate contains both hyaluronic acid (HA) and an integrin ligand (collagen I). HA is a major extracellular matrix component that is often up-regulated during wound healing and tumor growth. HA, together with collagen I, promotes hepatocellular carcinoma cell (Huh7) spreading on very soft substrates (300 Pa), resulting in morphology and motility similar to what these cells develop only on stiff substrates (>30 kPa) formed by polyacrylamide that contains collagen but not HA. The effect of HA requires turnover of Polyphosphoinositides and leads to the activation of Akt. The inhibition of Polyphosphoinositide turnover causes Huh7 cells and fibroblasts to decrease spreading and detach, whereas cells on stiffer substrates show almost no response. Traction force microscopy shows that the cell maintains a low strain energy and net contractile moment on HA substrates compared to stiff polyacrylamide substrates. Membrane tension measured by tether pulling is similar on soft HA and stiff polyacrylamide substrates. These results suggest that simultaneous signaling stimulated by HA and an integrin ligand can generate phosphoinositide-mediated signals to the cytoskeleton that reproduce those generated by high cellular tension
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electrostatic contribution to the surface pressure of charged monolayers containing Polyphosphoinositides
Biophysical Journal, 2008Co-Authors: Ilya Levental, Paul A Janmey, A CēbersAbstract:Structural and functional studies of lateral heterogeneity in biological membranes have underlined the importance of membrane organization in biological function. Most inquiries have focused on steric determinants of membrane organization, such as headgroup size and acyl-chain saturation. This manuscript reports a combination of theory and experiment that shows significant electrostatic contributions to surface pressures in monolayers of phospholipids where the charge spacing is smaller than the Bjerrum length. For molecules with steric cross sections typical of phospholipids in the cell membrane (;50 A ˚ 2 ), only Polyphosphoinositides achieve this threshold. The most abundant such lipid is phosphatidylinositol bisphosphate, which has between three and four charged groups at physiological conditions. Theory and experiment show that surface pressure increases linearly with phosphatidylinositol bisphosphate net charge and reveal crossing of high and low ionic strength pressure-area isotherms, due to opposing effects of ionic strength in compressed and expanded monolayers. Theory and experiment show that electrostatic effects are negligible for monolayers of univalent lipids, emphasizing the unique importance of electrostatic effects for lateral organization of Polyphosphoinositides. Quantitative differences between theory and experiment suggest that attractive interactions between Polyphosphoinositides, possibly mediated by hydrogen bonding, can lessen the effect of electrostatic repulsions.
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dissecting the gelsolin Polyphosphoinositide interaction and engineering of a Polyphosphoinositide sensitive gelsolin c terminal half protein
Journal of Molecular Biology, 2002Co-Authors: Wujing Xian, Paul A JanmeyAbstract:Gelsolin and other proteins in the villin/gelsolin family are regulated by Polyphosphoinositides (PPIs), and manipulation of cellular PIP(2) levels alters the structure of the actin cytoskeleton coincident with the dissociation of gelsolin-actin complexes. This work explores the structure-function relationship of the gelsolin-PPI interaction. Circular dichroism experiments show that upon binding to PPIs, the PPI-sensitive N-terminal half of gelsolin undergoes significant secondary and tertiary structural changes that do not occur in the structurally homologous but PPI-insensitive C-terminal half. Secondary structure modeling algorithms predict an alpha-helical conformation for one of the gelsolin PPI-binding sites, P2, which differs from the conformation of P2 in the structure of gelsolin determined by X-ray crystallography, whereas structure prediction of the C-terminal homolog of P2 agrees well with the X-ray crystallography structure. Simulation of a change to helical conformation for P2 using molecular modeling indicates that such a structural transition will destabilize the F-actin-binding sites in domain 2. A hypothesis is proposed that PPIs initiate conformational changes at the PPI-binding site(s) that destabilize the protein structure, and subsequently disrupt the actin-binding sites. To further evaluate the role of P2 in the gelsolin-PPI interaction, a Ct mutant P2Ct is constructed by inserting P2 in place of its C-terminal homologous site. P2Ct interacts with actin in the same way as the wild-type protein. In contrast to Ct, however, P2Ct interacts strongly with PPIs, and its monomeric actin-binding activity becomes regulated by PPIs. It is concluded that the P2 site is sufficient for PPI-sensitivity in gelsolin. Furthermore, the P2 site in P2Ct and the actin-binding sites of Ct do not overlap, suggesting that PPIs regulate actin binding of P2Ct through induction of structural changes, rather than through direct competition.
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cell permeant Polyphosphoinositide binding peptides that block cell motility and actin assembly
Journal of Biological Chemistry, 2001Co-Authors: Casey C Cunningham, John H Hartwig, Thomas P Stossel, Rolands Vegners, Robert Bucki, Makoto Funaki, Neha Korde, Paul A JanmeyAbstract:Polyphosphoinositides (PPIs) affect the localization and activities of many cellular constituents, including actin-modulating proteins. Several classes of polypeptide sequences, including pleckstrin homology domains, FYVE domains, and short linear sequences containing predominantly hydrophobic and cationic residues account for phosphoinositide binding by most such proteins. We report that a ten-residue peptide derived from the phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding region in segment 2 of gelsolin, when coupled to rhodamine B has potent PIP(2) binding activity in vitro; crosses the cell membrane of fibroblasts, platelets, melanoma cells, and neutrophils by a process not involving endocytosis; and blocks cell motility. This peptide derivative transiently disassembles actin filament structures in GFP-actin-expressing NIH3T3 fibroblasts and prevents thrombin- or chemotactic peptide-stimulated actin assembly in platelets and neutrophils, respectively, but does not block the initial [Ca(2+)] increase caused by these agonists. The blockage of actin assembly and motility is transient, and cells recover motility within an hour after their immobilization by 5-20 microm peptide. This class of reagents confirms the critical relation between inositol lipids and cytoskeletal structure and may be useful to probe the location and function of Polyphosphoinositides in vivo.
James W Putney - One of the best experts on this subject based on the ideXlab platform.
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Complex functions of phosphatidylinositol 4,5-bisphosphate in regulation of TRPC5 cation channels
Pflügers Archiv - European Journal of Physiology, 2009Co-Authors: Mohamed Trebak, Gary S Bird, Wayne I. Dehaven, Loic Lemonnier, Barbara J. Wedel, James W PutneyAbstract:The canonical transient receptor potential (TRPC) proteins have been recognized as key players in calcium entry pathways activated through phospholipase-C-coupled receptors. While it is clearly demonstrated that members of the TRPC3/6/7 subfamily are activated by diacylglycerol, the mechanism by which phospholipase C activates members of the TRPC1/4/5 subfamily remains a mystery. In this paper, we provide evidence for both negative and positive modulatory roles for membrane Polyphosphoinositides in the regulation of TRPC5 channels. Depletion of polyphosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (PIP_2) through inhibition of phosphatidylinositol 4-kinase activates calcium entry and membrane currents in TRPC5-expressing but not in TRPC3- or TRPC7-expressing cells. Inclusion of polyphosphatidylinositol 4-phosphate or PIP_2, but not phosphatidylinositol 3,4,5-trisphosphate, in the patch pipette inhibited TRPC5 currents. Paradoxically, depletion of PIP_2 with a directed 5-phosphatase strategy inhibited TRPC5. Furthermore, when the activity of single TRPC5 channels was examined in excised patches, the channels were robustly activated by PIP_2. These findings indicate complex functions for regulation of TRPC5 by PIP_2, and we propose that membrane Polyphosphoinositides may have at least two distinct functions in regulating TRPC5 channel activity.
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role of the phospholipase c inositol 1 4 5 trisphosphate pathway in calcium release activated calcium current and capacitative calcium entry
Journal of Biological Chemistry, 2001Co-Authors: Lisa M Broad, Franzjosef Braun, Jeanphilippe Lievremont, Gary S Bird, Tomohiro Kurosaki, James W PutneyAbstract:We investigated the putative roles of phospholipase C, Polyphosphoinositides, and inositol 1,4,5-trisphosphate (IP(3)) in capacitative calcium entry and calcium release-activated calcium current (I(crac)) in lacrimal acinar cells, rat basophilic leukemia cells, and DT40 B-lymphocytes. Inhibition of phospholipase C with blocked calcium entry and I(crac) activation whether in response to a phospholipase C-coupled agonist or to calcium store depletion with thapsigargin. Run-down of cellular Polyphosphoinositides by concentrations of wortmannin that block phosphatidylinositol 4-kinase completely blocked calcium entry and I(crac). The membrane-permeant IP(3) receptor inhibitor, 2-aminoethoxydiphenyl borane, blocked both capacitative calcium entry and I(crac). However, it is likely that 2-aminoethoxydiphenyl borane does not inhibit through an action on the IP(3) receptor because the drug was equally effective in wild-type DT40 B-cells and in DT40 B-cells whose genes for all three IP(3) receptors had been disrupted. Intracellular application of another potent IP(3) receptor antagonist, heparin, failed to inhibit activation of I(crac). Finally, the inhibition of I(crac) activation by or wortmannin was not reversed or prevented by direct intracellular application of IP(3). These findings indicate a requirement for phospholipase C and for Polyphosphoinositides for activation of capacitative calcium entry. However, the results call into question the previously suggested roles of IP(3) and IP(3) receptor in this mechanism, at least in these particular cell types.
Glenn D Prestwich - One of the best experts on this subject based on the ideXlab platform.
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specific interaction of golgi coatomer protein α cop with phosphatidylinositol 3 4 5 trisphosphate
Journal of Biological Chemistry, 1998Co-Authors: Anu Chaudhary, Oliver Thum, Adam A Profit, Loice H Jeyakumar, Sidney Fleischer, Glenn D PrestwichAbstract:Abstract The phosphoinositide binding selectivity of Golgi coatomer COPI polypeptides was examined using photoaffinity analogs of the soluble inositol polyphosphates Ins(1,4,5)P3, Ins(1,3,4,5)P4, and InsP6, and of the Polyphosphoinositides PtdIns(3,4,5)P3, PtdIns(4,5)P2, and PtdIns(3,4)P2. Highly selective Ins(1,3,4,5)P4-displaceable photocovalent modification of the α-COP subunit was observed with ap-benzoyldihydrocinnamide (BZDC)-containing probe, [3H]BZDC-Ins(1,3,4,5)P4. A more highly phosphorylated probe, [3H]BZDC-InsP6probe labeled six of the seven subunits, with only β, β′, δ, and e-COP showing competitive displacement by excess InsP6. Importantly, [3H]BZDC-triester-PtdIns(3,4,5)P3, the lipid with the same phosphorylation pattern as Ins(1,3,4,5)P4, showed specific, PtdIns(3,4,5)P3-displaceable labeling of only α-COP. Labeling by the PtdIns(4,5)P2 and PtdIns(3,4)P2 photoaffinity probes was less intense and showed no discrimination based on PtdInsPn ligand. Thus, both the D-3 and D-5 phosphates are critical for the α-COP-PtdIns(3,4,5)P3 interaction, suggesting an important role for this Polyphosphoinositide in vesicular trafficking.
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Phosphatidylinositol-4-phosphate 5-kinase isozymes catalyze the synthesis of 3-phosphate-containing phosphatidylinositol signaling molecules.
Journal of Biological Chemistry, 1997Co-Authors: Xiaoling Zhang, Philip W. Majerus, Joost C. Loijens, Igor V. Boronenkov, F. Anderson Norris, Oliver Thum, Gregory J Parker, Glenn D Prestwich, Jian Chen, Richard A AndersonAbstract:Abstract Phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) utilize phosphatidylinositols containing D-3-position phosphates as substrates to form phosphatidylinositol 3,4-bisphosphate. In addition, type I PIP5Ks phosphorylate phosphatidylinositol 3,4-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate, while type II kinases have less activity toward this substrate. Remarkably, these kinases can convert phosphatidylinositol 3-phosphate to phosphatidylinositol 3,4,5-trisphosphate in a concerted reaction. Kinase activities toward the 3-position phosphoinositides are comparable with those seen with phosphatidylinositol 4-phosphate as the substrate. Therefore, the PIP5Ks can synthesize phosphatidylinositol 4,5-bisphosphate and two 3-phosphate-containing Polyphosphoinositides. These unexpected activities position the PIP5Ks as potential participants in the generation of all Polyphosphoinositide signaling molecules.
Linda J. Pike - One of the best experts on this subject based on the ideXlab platform.
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phosphoinositides and phosphoinositide utilizing enzymes in detergent insoluble lipid domains
Molecular Biology of the Cell, 1996Co-Authors: H R Hope, Linda J. PikeAbstract:Recent evidence has implicated caveolae/DIGs in various aspects of signal transduction, a process in which Polyphosphoinositides play a central role. We therefore undertook a study to determine the distribution of phosphoinositides and the enzymes that utilize them in these detergent-insoluble domains. We report here that the Polyphosphoinositide phosphatase, but not several other phosphoinositide-utilizing enzymes, is highly enriched in a low density, Triton-insoluble membrane fraction that contains caveolin. This fraction is also enriched in Polyphosphoinositides, containing approximately one-fifth of the total cellular phosphatidylinositol (4,5)P2. Treatment of cells with the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), did not alter the distribution of Polyphosphoinositides or the Polyphosphoinositide phosphatase. However, PMA treatment did lead to a decrease in the mitogen-activated protein kinase and actin present in these domains. PMA also induced the recruitment of protein kinase C alpha to the caveolae/DIGs fraction. These findings suggest that Polyphosphoinositides, the Polyphosphoinositide phosphatase and protein kinase C play an important role in the structure or function of detergent-insoluble membrane domains.
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purification and characterization of a Polyphosphoinositide phosphatase from rat brain
Journal of Biological Chemistry, 1994Co-Authors: H M R Hope, Linda J. PikeAbstract:A novel membrane-bound Polyphosphoinositide phosphatase has been purified 7700-fold from rat brain. A combination of gel filtration chromatography and SDS-polyacrylamide gel electrophoresis indicated that the enzyme is a monomer with a molecular weight of 85,000-90,000. Biochemical analysis of the Polyphosphoinositide phosphatase demonstrated that the enzyme utilizes phosphatidylinositol(4)phosphate (PtdIns(4)P), phosphatidylinositol(3)phosphate (PtdIns(3)P), and phosphatidylinositol (4,5)bisphosphate (PtdIns(4,5)P2) as substrates. In the case of PtdIns(4,5)P2, the substrate is doubly dephosphorylated to yield PtdIns. The apparent Km values for PtdIns(4)P and PtdIns(4,5)P2 are 45 and 5 microM, respectively. Inositol(1,4)bisphosphate and inositol(1,4,5)trisphosphate neither serve as direct substrates for the Polyphosphoinositide phosphatase nor inhibit its activity even at concentrations as high as 100 microM. Thus, the substrate specificity of the Polyphosphoinositide phosphatase is distinct from that of previously identified phosphatases that utilize both inositol phospholipids and soluble inositol phosphates as substrates. The ability of the Polyphosphoinositide phosphatase to hydrolyze phosphate from the 3-, 4-, or 5-position of the inositol ring suggests that this enzyme may play a key role in maintaining homeostasis among all forms of Polyphosphoinositides.
Richard A Anderson - One of the best experts on this subject based on the ideXlab platform.
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Phosphatidylinositol-4-phosphate 5-kinase isozymes catalyze the synthesis of 3-phosphate-containing phosphatidylinositol signaling molecules.
Journal of Biological Chemistry, 1997Co-Authors: Xiaoling Zhang, Philip W. Majerus, Joost C. Loijens, Igor V. Boronenkov, F. Anderson Norris, Oliver Thum, Gregory J Parker, Glenn D Prestwich, Jian Chen, Richard A AndersonAbstract:Abstract Phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) utilize phosphatidylinositols containing D-3-position phosphates as substrates to form phosphatidylinositol 3,4-bisphosphate. In addition, type I PIP5Ks phosphorylate phosphatidylinositol 3,4-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate, while type II kinases have less activity toward this substrate. Remarkably, these kinases can convert phosphatidylinositol 3-phosphate to phosphatidylinositol 3,4,5-trisphosphate in a concerted reaction. Kinase activities toward the 3-position phosphoinositides are comparable with those seen with phosphatidylinositol 4-phosphate as the substrate. Therefore, the PIP5Ks can synthesize phosphatidylinositol 4,5-bisphosphate and two 3-phosphate-containing Polyphosphoinositides. These unexpected activities position the PIP5Ks as potential participants in the generation of all Polyphosphoinositide signaling molecules.