Laurdan fluorescence senses mechanical strain in the lipid bilayer membrane

The precise molecular mechanisms by which cells transduce a mechanical stimulus into an intracellular biochemical response have not yet been established. Here, we show for the first time that the fluorescence emission of an environment-sensitive membrane probe Laurdan is modulated by mechanical strain of the lipid bilayer membrane. We have measured fluorescence emission of Laurdan in phospholipid vesicles of 30, 50, and 100 nm diameter to show that osmotically induced membrane tension leads to an increase in polarity (hydration depth) of the phospholipid bilayer interior. Our data indicate that the general polarization of Laurdan emission is linearly dependent on membrane tension. We also show that higher membrane curvature leads to higher hydration levels. We anticipate that the proposed method will facilitate future studies of mechanically induced changes in physical properties of lipid bilayer environment both in vitro and in vivo.     

PTH1 Receptor Is Involved in Mediating Cellular Response to Long-Chain Polyunsaturated Fatty Acids

The molecular pathways by which long chain polyunsaturated fatty acids (LCPUFA) influence skeletal health remain elusive. Both LCPUFA and parathyroid hormone type 1 receptor (PTH1R) are known to be involved in bone metabolism while any direct link between the two is yet to be established. Here we report that LCPUFA are capable of direct, PTH1R dependent activation of extracellular ligand-regulated kinases (ERK). From a wide range of fatty acids studied, varying in chain length, saturation, and position of double bonds, eicosapentaenoic (EPA) and docosahexaenoic fatty acids (DHA) caused the highest ERK phosphorylation. Moreover, EPA potentiated the effect of parathyroid hormone (PTH(1–34)) in a superagonistic manner. EPA or DHA dependent ERK phosphorylation was inhibited by the PTH1R antagonist and by knockdown of PTH1R. Inhibition of PTH1R downstream signaling molecules, protein kinases A (PKA) and C (PKC), reduced EPA and DHA dependent ERK phosphorylation indicating that fatty acids predominantly activate G-protein pathway and not the β-arrestin pathway. Using picosecond time-resolved fluorescence microscopy and a genetically engineered PTH1R sensor (PTH-CC), we detected conformational responses to EPA similar to those caused by PTH(1–34). PTH1R antagonist blocked the EPA induced conformational response of the PTH-CC. Competitive binding studies using fluorescence anisotropy technique showed that EPA and DHA competitively bind to and alter the affinity of PTH1 receptor to PTH(1–34) leading to a superagonistic response. Finally, we showed that EPA stimulates protein kinase B (Akt) phosphorylation in a PTH1R-dependent manner and affects the osteoblast survival pathway, by inhibiting glucocorticoid-induced cell death. Our findings demonstrate for the first time that LCPUFAs, EPA and DHA, can activate PTH1R receptor at nanomolar concentrations and consequently provide a putative molecular mechanism for the action of fatty acids in bone.     

Elastic light scattering from single cells: orientational dynamics in optical trap

Light-scattering diagrams (phase functions) from single living cells and beads suspended in an optical trap were recorded with 30-ms time resolution. The intensity of the scattered light was recorded over an angular range of 0.5-179.5 degrees using an optical setup based on an elliptical mirror and rotating aperture. Experiments revealed that light-scattering diagrams from biological cells exhibit significant and complex time dependence. We have attributed this dependence to the cell's orientational dynamics within the trap. We have also used experimentally measured phase function information to calculate the time dependence of the optical radiation pressure force on the trapped particle and show how it changes depending on the orientation of the particle. Relevance of these experiments to potential improvement in the sensitivity of label-free flow cytometry is discussed.     

Effect of membrane tension on the electric field and dipole potential of lipid bilayer membrane

The dipole potential of lipid bilayer membrane controls the difference in permeability of the membrane to oppositely charged ions. We have combined molecular dynamics (MD) simulations and experimental studies to determine changes in electric field and electrostatic potential of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer in response to applied membrane tension. MD simulations based on CHARMM36 force field showed that electrostatic potential of DOPC bilayer decreases by ~45mV in the physiologically relevant range of membrane tension values (0 to 15dyn/cm). The electrostatic field exhibits a peak (~0.8×10(9)V/m) near the water/lipid interface which shifts by 0.9? towards the bilayer center at 15dyn/cm. Maximum membrane tension of 15dyn/cm caused 6.4% increase in area per lipid, 4.7% decrease in bilayer thickness and 1.4% increase in the volume of the bilayer. Dipole-potential sensitive fluorescent probes were used to detect membrane tension induced changes in DOPC vesicles exposed to osmotic stress. Experiments confirmed that dipole potential of DOPC bilayer decreases at higher membrane tensions. These results are suggestive of a potentially new mechanosensing mechanism by which mechanically induced structural changes in the lipid bilayer membrane could modulate the function of membrane proteins by altering electrostatic interactions and energetics of protein conformational states.     

Counter-propagating optical trapping system for size and refractive index measurement of microparticles

We propose and demonstrate a novel approach to measure the size and refractive index of microparticles based on two beam optical trapping, where forward scattered light is detected to give information about the particle. The counter-propagating optical trap measurement (COTM) system exploits the capability of optical traps to measure pico-Newton forces for microparticles' refractive index and size characterization. Different from the current best technique for microparticles' refractive index measurement, refractometry, a bulk technique requiring changing the fluid composition of the sample, our optical trap technique works with any transparent fluid and enables single particle analysis without the use of biological markers. A ray-optics model is used to explore the physical operation of the COTM system, predict system performance and aid system design. Experiments demonstrate the accuracy of refractive index measurement of Deltan=0.013 and size measurement of 3% of diameter with 2% standard deviation. Present performance is instrumentation limited, and a potential improvement by more than two orders of magnitude can be expected in the future. With further development in parallelism and miniaturization, the system offers advantages for cell manipulation and bioanalysis compatible with lab-on-a-chip systems.     

Use of moving optical gradient fields for analysis of apoptotic cellular responses in a chronic myeloid leukemia cell model

To facilitate quantitation of cellular apoptotic responses to various antineoplastic agents, a laser-based technology, Optophoresis, has been developed to provide analysis of cells without any need for labeling or cell processing. Optophoresis is defined as the analysis of the motion of cells, where the motion is either induced or modified by a moving optical gradient field, which produces radiation pressure forces on the cells in an aqueous suspension. Quantitation of the induced motion provides a basis for distinguishing one population of cells from another. One Optophoretic technique, Fast Scan, measures the distribution of distances traversed by a population of cells when exposed to a fast-moving optical gradient. Fast Scan was validated using a cell-based model of chronic myeloid leukemia treated with Gleevec, a specific inhibitor of aberrant Bcr-Abl protein kinase. The Optophoretic measurements were quantitatively comparable to reference assays with regard to drug selectivity and potency and to target specificity, demonstrating the suitability of this technology for pharmaceutical and clinical applications.     

Effect of membrane tension on the physical properties of DOPC lipid bilayer membrane

Molecular dynamics simulations of a dioleoylphosphocholine (DOPC) lipid bilayer were performed to explore its mechanosensitivity. Variations in the bilayer properties, such as area per lipid, volume, thickness, hydration depth (HD), hydration thickness (HT), lateral diffusion coefficient, and changes in lipid structural order were computed in the membrane tension range 0 to 15dyn/cm. We determined that an increase in membrane tension results in a decrease in the bilayer thickness and HD of ~5% and ~5.7% respectively, whereas area per lipid, volume, and HT/HD increased by 6.8%, 2.4%, and 5% respectively. The changes in lipid conformation and orientation were characterized using orientational (S(2)) and deuterium (S(CD)) order parameters. Upon increase of membrane tension both order parameters indicated an increase in lipid disorder by 10-20%, mostly in the tail end region of the hydrophobic chains. The effect of membrane tension on lipid lateral diffusion in the DOPC bilayer was analyzed on three different time scales corresponding to inertial motion, anomalous diffusion and normal diffusion. The results showed that lateral diffusion of lipid molecules is anomalous in nature due to the non-exponential distribution of waiting times. The anomalous and normal diffusion coefficients increased by 20% and 52% when the membrane tension changed from 0 to 15dyn/cm, respectively. In conclusion, our studies showed that membrane tension causes relatively significant changes in the area per lipid, volume, polarity, membrane thickness, and fluidity of the membrane suggesting multiple mechanisms by which mechanical perturbation of the membrane could trigger mechanosensitive response in cells.