Cell Membranes Resist Flow.

The fluid-mosaic model posits a liquid-like plasma membrane, which can flow in response to tension gradients. It is widely assumed that membrane flow transmits local changes in membrane tension across the cell in milliseconds, mediating long-range signaling. Here, we show that propagation of membrane tension occurs quickly in cell-attached blebs but is largely suppressed in intact cells. The failure of tension to propagate in cells is explained by a fluid dynamical model that incorporates the flow resistance from cytoskeleton-bound transmembrane proteins. Perturbations to tension propagate diffusively, with a diffusion coefficient Dσ ∼0.024 µm2/s in HeLa cells. In primary endothelial cells, local increases in membrane tension lead only to local activation of mechanosensitive ion channels and to local vesicle fusion. Thus, membrane tension is not a mediator of long-range intracellular signaling, but local variations in tension mediate distinct processes in sub-cellular domains.

Effect of H-Bond Donor Lipids on Phosphatidylinositol-3,4,5-Trisphosphate Ionization and Clustering.

The phosphoinositide, phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3), is a key signaling lipid in the inner leaflet of the cell plasma membrane, regulating diverse signaling pathways including cell growth and migration. In this study we investigate the impact of the hydrogen-bond donor lipids phosphatidylethanolamine (PE) and phosphatidylinositol (PI) on the charge and phase behavior of PI(3,4,5)P3. PE and PI can interact with PI(3,4,5)P3 through hydrogen-bond formation, leading to altered ionization behavior and charge distribution within the PI(3,4,5)P3 headgroup. We quantify the altered PI(3,4,5)P3 ionization behavior using a multistate ionization model to obtain micro-pKa values for the ionization of each phosphate group. The presence of PE leads to a decrease in the pKa values for the initial deprotonation of PI(3,4,5)P3, which describes the removal of the first proton of the three protons remaining at the phosphomonoester groups at pH 4.0. The decrease in these micro-pKa values thus leads to a higher charge at low pH. Additionally, the charge distribution changes lead to increased charge on the 3- and 5-phosphates. In the presence of PI, the final deprotonation of PI(3,4,5)P3 is delayed, leading to a lower charge at high pH. This is due to a combination of hydrogen-bond formation between PI and PI(3,4,5)P3, and increased surface charge due to the addition of the negatively charged PI. The interaction between PI and PI(3,4,5)P3 leads to the formation of PI and PI(3,4,5)P3-enriched domains within the membrane. These domains may have a critical impact on PI(3,4,5)P3-signaling. We also reevaluate results for all phosphatidylinositol bisphosphates as well as for PI(4,5)P2 in complex lipid mixtures with the multistate ionization model.

Dendrimersomes Exhibit Lamellar-to-Sponge Phase Transitions.

Lamellar to nonlamellar membrane shape transitions play essential roles in key cellular processes, such as membrane fusion and fission, and occur in response to external stimuli, including drug treatment and heat. A subset of these transitions can be modeled by means of thermally inducible amphiphile assemblies. We previously reported on mixtures of hydrogenated, fluorinated, and hybrid Janus dendrimers (JDs) that self-assemble into complex dendrimersomes (DMSs), including dumbbells, and serve as promising models for understanding the complexity of biological membranes. Here we show, by means of a variety of complementary techniques, that DMSs formed by single JDs or by mixtures of JDs undergo a thermally induced lamellar-to-sponge transition. Consistent with the formation of a three-dimensional bilayer network, we show that DMSs become more permeable to water-soluble fluorophores after transitioning to the sponge phase. These DMSs may be useful not only in modeling isotropic membrane rearrangements of biological systems but also in drug delivery since nonlamellar delivery vehicles can promote endosomal disruption and cargo release.

Probing lipid membrane bending mechanics using gold nanorod tracking.

Lipid bilayer membranes undergo rapid bending undulations with wavelengths from tens of nanometers to tens of microns due to thermal fluctuations. Here, we probe such undulations and the membranes' mechanics by measuring the time-varying orientation of single gold nanorods (GNRs) adhered to the membrane, using high-speed dark field microscopy. In a lipid vesicle, such measurements allow the determination of the membrane's viscosity, bending rigidity, and tension as well as the friction coefficient for sliding of the monolayers over one another. The in-plane rotation of the GNR is hindered by undulations in a tension dependent manner, consistent with simulations. The motion of single GNRs adhered to the plasma membrane of living cultured cells similarly reveals the membrane's complex physics and coupling to the cell's actomyosin cortex.

The Electrostatic Basis of Diacylglycerol Pyrophosphate-Protein Interaction.

Diacylglycerol pyrophosphate (DGPP) is an anionic phospholipid formed in plants, yeast, and parasites under multiple stress stimuli. It is synthesized by the phosphorylation action of phosphatidic acid (PA) kinase on phosphatidic acid, a signaling lipid with multifunctional properties. PA functions in the membrane through the interaction of its negatively charged phosphomonoester headgroup with positively charged proteins and ions. DGPP, like PA, can interact electrostatically via the electrostatic-hydrogen bond switch mechanism but differs from PA in its overall charge and shape. The formation of DGPP from PA alters the physicochemical properties as well as the structural dynamics of the membrane. This potentially impacts the molecular and ionic binding of cationic proteins and ions with the DGPP enriched membrane. However, the results of these important interactions in the stress response and in DGPP's overall intracellular function is unknown. Here, using 31P MAS NMR, we analyze the effect of the interaction of low DGPP concentrations in model membranes with the peptides KALP23 and WALP23, which are flanked by positively charged Lysine and neutral Tryptophan residues, respectively. Our results show a significant effect of KALP23 on the charge of DGPP as compared to WALP23. There was, however, no significant effect on the charge of the phosphomonoester of DGPP due to the interaction with positively charged lipids, dioleoyl trimethylammonium propane (DOTAP) and dioleoyl ethyl-phosphatidylcholine (EtPC). Divalent calcium and magnesium cations induce deprotonation of the DGPP headgroup but showed no noticeable differences on DGPP's charge. Our results lead to a novel model for DGPP-protein interaction.

Soft Hyaluronic Gels Promote Cell Spreading, Stress Fibers, Focal Adhesion, and Membrane Tension by Phosphoinositide Signaling, Not Traction Force.

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.

Phosphatidylinositol-4,5-bisphosphate ionization in the presence of cholesterol, calcium or magnesium ions.

Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) is an important signaling lipid and plays a crucial role in a wide variety of cellular processes by interacting with protein targets and localizing proteins at the plasma membrane. These interactions are strongly influenced by the lateral distribution of PI(4,5)P2 as well as its ionization state. The characterization of the PI(4,5)P2 ionization state provides important information about how PI(4,5)P2 interacts with other membrane resident or associated chemical species. In this study we have used solid-state MAS (31)P NMR to investigate the interactions of PI(4,5)P2 with potential cluster promoting agents, divalent cations and cholesterol. Both Ca(2+) and cholesterol were found previously to promote formation of local PI(4,5)P2 clusters in vitro. The NMR approach allows us to probe independently the ionization state of PI(4,5)P2 two phosphomonoester groups. We investigated mixed phosphatidylcholine (PC)/PI(4,5)P2 multilamellar vesicles in the presence of micro and millimolar concentrations of Ca(2+) and Mg(2+). We found that both cations lead to an increased downfield chemical shift of the PI(4,5)P2 phosphomonoester peaks, indicating an increased ionization in the presence of the divalent cations. Ca(2+) has a much larger effect on PI(4,5)P2 as compared to Mg(2+) at similar concentrations. Physiological concentrations of Ca(2+) are significantly lower than those found for Mg(2+) and the comparison of the PI(4,5)P2 ionization in the presence of Ca(2+) and Mg(2+) at physiological concentrations resulted in similar charges of the phosphomonoester groups for both cations. PI(4,5)P2 was also examined with vesicles containing cholesterol since cholesterol has been shown to promote PI(4,5)P2 clustering. In the presence of 40 mol% cholesterol, the PI(4,5)P2 phosphomonoester (31)P NMR peaks shifted slightly downfield, indicating a small increase in charge. Previously published data suggest that PI(4,5)P2 is capable of forming an intra- and intermolecular hydrogen bond network, which leads to a reduction of the charge at the phosphomonoester groups through dissipation of the charge across the bilayer/water interface. We hypothesize that cholesterol participates in this intermolecular hydrogen bond network, resulting in a stabilization of PI(4,5)P2 enriched domains due an increased spacing between the PI(4,5)P2 headgroup. We also examined the cumulative effects of cholesterol combined with the divalent cations, phosphatidylethanolamine (PE), and phosphatidylinositol (PI), separately. The combination of cholesterol and divalent cations results in an additive effect on PI(4,5)P2 ionization, while the effect of cholesterol on PI(4,5)P2 ionization is reduced in the presence of PE or PI.

Ionization behavior of polyphosphoinositides determined via the preparation of pH titration curves using solid-state 31P NMR.

Detailed knowledge of the degree of ionization of lipid titratable groups is important for the evaluation of protein-lipid and lipid-lipid interactions. The degree of ionization is commonly evaluated by acid-base titration, but for lipids localized in a multicomponent membrane interface this is not a suitable technique. For phosphomonoester-containing lipids such as the polyphosphoinositides, phosphatidic acid, and ceramide-1-phosphate, this is more conveniently accomplished by (31)P NMR. Here, we describe a solid-state (31)P NMR procedure to construct pH titration curves to determine the degree of ionization of phosphomonoester groups in polyphosphoinositides. This procedure can also be used, with suitable sample preparation conditions, for other important signaling lipids. Access to a solid-state, i.e., magic angle spinning, capable NMR spectrometer is assumed. The procedures described here are valid for a Bruker instrument, but can be adapted for other spectrometers as needed.

Phosphatidylinositol-4,5-bisphosphate ionization and domain formation in the presence of lipids with hydrogen bond donor capabilities.

Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) is an important lipidic signaling molecule that is involved in a broad range of cellular processes. Its interaction with proteins and its lateral distribution are governed by the ionization state of the phosphomonoester groups and its ability to form intra- and intermolecular hydrogen bonds. In this study we have investigated the ionization state of PI(4,5)P(2) in ternary lipid vesicle systems that contain in addition to PI(4,5)P(2) and phosphatidylcholine (PC) either phosphatidylethanolamine (PE), phosphatidylserine (PS) or phosphatidylinositol (PI). In the presence of PE we find an increased ionization of PI(4,5)P(2), which can be attributed to increased deprotonation due to hydrogen bond formation between PE and the PI(4,5)P(2) phosphomonoester groups. However, the effect of PE on PI(4,5)P(2) ionization is significantly smaller than it had been found previously for phosphatidic acid in the presence of PE (Kooijman et al., 2005). The reduced impact of PE on PI(4,5)P(2) ionization can be attributed to competing intramolecular hydrogen bond formation between the phosphomonoester groups and neighboring hydroxyl groups. It is noteworthy that the presence of PE affects more strongly the ionization of the 5-phosphate group than that of the 4-phosphate, suggesting that the interaction of PE with the 5-phosphate is stronger. In PI(4,5)P(2)/PS/PC lipid vesicles, the presence of PS was expected to yield an increased protonation of the PI(4,5)P(2) phosphomonoester groups due to a decreased interfacial pH as a result of the increased negative interfacial charge. However, the effect of PS on PI(4,5)P(2) ionization is only minor, potentially suggesting that PS and PI(4,5)P(2) are demixed. The PI(4,5)P(2)/PI/PC vesicle system was characterized by a surprising mixing behavior that has potentially far reaching consequences: fluorescence microscopy measurements of giant unilammellar vesicles composed of PI(4,5)P(2)/PI/PC at physiological concentrations show that PI and PI(4,5)P(2) form macroscopic, fluid phase domains in contact with a fluid PC rich phase (fluid/fluid demixing). Despite the fact that PI and PI(4,5)P(2) co-localize, the effect of PI on PI(4,5)P(2) ionization behavior is only noticeable above pH 7. Apparently two opposing effects lead to the observed behavior: Due to the presence of the anionic PI, the interfacial pH drops, which is expected to lead to an enhanced protonation of the PI(4,5)P(2) phosphomonoester groups. In turn, hydrogen bond formation between PI and PI(4,5)P(2) would lead to the opposite, i.e. increased deprotonation of the phosphomonoester group. Apparently these two effects compensate each other for pH values smaller than about 7, while for higher pH values the increased interfacial pH in the presence of PI has a stronger impact than PI/PI(4,5)P(2) hydrogen bond formation. The cooperative formation of PI/PI(4,5)P(2) mixed domains has potentially important ramifications for the spatial organization of phosphoinositide mediated signaling events.