Atomic force microscopy and light scattering of small unilamellar actin-containing liposomes

Three-dimensional networks of filamentous actin (F-actin) encapsulated inside phosphatidylcholine liposomes are currently being used in an effort to model the cytoskeleton and plasma membrane of eukaryotic cells. In this article, unilamellar lipid vesicles consisting of egg yolk-derived phosphatidylcholine encapsulating monomeric actin (G-actin) were made via extrusion in low ionic strength buffer (G-buffer). Vesicle shape and structure in these dispersions was studied using a combination of fluid-tapping atomic force microscopy, and multiangle static light scattering. After subjecting the liposome dispersion to high ionic strength polymerization buffer (F-buffer) containing K(+) ions, atomic force microscopy imaging and light scattering of these liposomes indicated the formation of specialized structures, including an overall liposome structure transformation from spherical to torus, disk-shaped geometries and tubular assemblies. Several atomic force microscopy control measurements were made to ascertain that the specialized structures formed were not due to free G-actin and F-actin self-assembling on the sample surface, plain liposomes exposed to G- and F-buffer, or liposomes encapsulating G-actin. Liposomes encapsulating G-actin assumed mostly thin disk shapes and some large irregularly shaped aggregates. In contrast, liposomes encapsulating polymerized actin assumed mostly torus or disk shapes along with some high aspect ratio tubular structures.     

Formation and maintenance of tubular membrane projections: experiments and numerical calculations

To study the mechanical properties of lipid membranes, we manipulated liposomes by using a system comprising polystyrene beads and laser tweezers, and measured the force required to transform their shapes. When two beads pushed the membrane from inside, spherical liposomes transformed into a lemon-shape. Then a discontinuous shape transformation occurred to form a membrane tube from either end of the liposomes, and the force dropped drastically. We analyzed these processes using a mathematical model based on the bending elasticity of the membranes. Numerical calculations showed that when the bead size was taken into account, the model reproduced both the liposomal shape transformation and the force-extension relation. This result suggests that the size of the beads is responsible for the existence of a force barrier for the tube formation.     

Membrane tether formation from outer hair cells with optical tweezers

Optical tweezers were used to characterize the mechanical properties of the outer hair cell (OHC) plasma membrane by pulling tethers with 4.5-microm polystyrene beads. Tether formation force and tether force were measured in static and dynamic conditions. A greater force was required for tether formations from OHC lateral wall (499 +/- 152 pN) than from OHC basal end (142 +/- 49 pN). The difference in the force required to pull tethers is consistent with an extensive cytoskeletal framework associated with the lateral wall known as the cortical lattice. The apparent plasma membrane stiffness, estimated under the static conditions by measuring tether force at different tether length, was 3.71 pN/microm for OHC lateral wall and 4.57 pN/microm for OHC basal end. The effective membrane viscosity was measured by pulling tethers at different rates while continuously recording the tether force, and estimated in the range of 2.39 to 5.25 pN x s/microm. The viscous force most likely results from the viscous interactions between plasma membrane lipids and the OHC cortical lattice and/or integral membrane proteins. The information these studies provide on the mechanical properties of the OHC lateral wall is important for understanding the mechanism of OHC electromotility.     

Effect of temperature on tether extraction, surface protrusion, and cortical tension of human neutrophils

Neutrophil rolling on endothelial cells, the initial stage of its migrational journey to a site of inflammation, is facilitated by tether extraction and surface protrusion. Both phenomena have been studied extensively at room temperature, which is considerably lower than human body temperature. It is known that temperature greatly affects cellular mechanical properties such as viscosity. Therefore, we carried out tether extraction, surface protrusion, and cortical tension experiments at 37 degrees C with the micropipette aspiration technique. The experimental temperature was elevated using a custom-designed microscope chamber for the micropipette aspiration technique. To evaluate the constant temperature assumption in our experiments, the temperature distribution in the whole chamber was computed with finite element simulation. Our simulation results showed that temperature variation around the location where our experiments were performed was less than 0.2 degrees C. For tether extraction at 37 degrees C, the threshold force required to pull a tether (40 pN) was not statistically different from the value at room temperature (51 pN), whereas the effective viscosity (0.75 pN.s/microm) decreased significantly from the value at room temperature (1.5 pN.s/microm). Surface protrusion, which was modeled as a linear deformation, had a slightly smaller spring constant at 37 degrees C (40 pN/microm) than it did at room temperature (56 pN/microm). However, the cortical tension at 37 degrees C (5.7+/-2.2 pN/microm) was substantially smaller than that at room temperature (23+/-8 pN/microm). These data clearly suggest that neutrophils roll differently at body temperature than they do at room temperature by having distinct mechanical responses to shear stress of blood flow.     

Characteristics of a membrane reservoir buffering membrane tension.

When membrane-attached beads are pulled vertically by a laser tweezers, a membrane tube of constant diameter (tether) is formed. We found that the force on the bead (tether force) did not depend on tether length over a wide range of tether lengths, which indicates that a previously unidentified reservoir of membrane and not stretch of the plasma membrane provides the tether membrane. Plots of tether force vs. tether length have an initial phase, an elongation phase, and an exponential phase. During the major elongation phase, tether force is constant, buffered by the "membrane reservoir." Finally, there is an abrupt exponential rise in force that brings the tether out of the trap, indicating depletion of the membrane reservoir. In chick embryo fibroblasts and 3T3 fibroblasts, the maximum tether lengths that can be pulled at a velocity of 4 microm/s are 5.1 +/- 0. 3 and 5.0 +/- 0.2 microm, respectively. To examine the importance of the actin cytoskeleton, we treated cells with cytochalasin B or D and found that the tether lengths increased dramatically to 13.8 +/- 0.8 and 12.0 +/- 0.7 microm, respectively. Similarly, treatment of the cells with colchicine and nocodazole results in more than a twofold increase in tether length. We found that elevation of membrane tension (through osmotic pressure, a long-term elevation of tether force, or a number of transitory increases) increased reservoir size over the whole cell. Using a tracking system to hold tether force on the bead constant near its maximal length in the exponential phase, the rate of elongation of the tethers was measured as a function of tether force (membrane tension). The rate of elongation of tethers was linearly dependent on the tether force and reflected an increase in size of the reservoir. Increases in the reservoir caused by tension increases on one side of the cell caused increases in reservoir size on the other side of the cell. Thus, we suggest that cells maintain a plasma membrane reservoir to buffer against changes in membrane tension and that the reservoir is increased with membrane tension or disruption of the cytoskeleton.     

The effects of liposome size and surface charge on liposome-mediated delivery of methotrexate-gamma-aspartate to cells in vitro

We have studied the liposome-mediated delivery of methotrexate-gamma-aspartate to five cell lines. The sensitivity of the cells to encapsulated drug varies widely in accordance with their ability to take up the liposomes. CV1-P cells can be 150-times more sensitive to encapsulated methotrexate-gamma-aspartate than to free drug, while AKR/J SL2 cells are only twice as sensitive to the encapsulated drug. Negatively-charged liposomes are much more efficient for delivery than are neutral liposomes, and cholesterol is an essential component of the liposome membrane for optimal drug delivery. The optimal liposome size for drug delivery is 0.1 micron, although the amount of cell-associated lipid is the same for all liposome sizes. The effect of the encapsulated drug is inhibited by NH4Cl, suggesting an endocytic mechanism for delivery. The potency of the encapsulated drug is not affected by wide variations in the drug: lipid ratio.     

Morphological and Topological Transformation of Membrane Vesicles

Liposomes are micro-compartments made of lipid bilayer membranes withcharacteristics quite similar to those of biological membranes. To formartificial cell-like structures, we generated liposomes that containedsubunit proteins of cytoskeletons: tubulin or actin. Spherical liposomeswere transformed into bipolar or cell-like shapes by mechanical forcesgenerated by the polymerization of encapsulated subunits of microtubules.Disk- or dumbbell-shaped liposomes were developed by the polymerizationof encapsulated actin. Dynamic processes of morphological transformationsof liposomes were visualized by high intensity dark-field lightmicroscopy.Topological changes, such as fusion and division of membrane vesicles,play an essential role in cellular activities. To investigate themechanism of these processes, we visualized in real time the liposomesundergoing topological transformation. A variety of novel topologicaltransformations were found, including the opening-up of liposomes and thedirect expulsion of inner vesicles.     

Characterization of the surfaces generated by liposome binding to the modified dextran matrix of a surface plasmon resonance sensor chip

The dextran matrix of a surface plasmon resonance (SPR) sensor chip modified with hydrophobic residues (BIAcore sensor chip L1) provides an ideal substrate for liposome adsorption. Liposomes of different lipid compositions are captured on the sensor chips by inserting these residues into the liposome membrane, thereby generating stable lipid surfaces. To gain a more detailed understanding of these surfaces, and to prove whether the liposomes stay on the matrix as single particles or form a continuous lipid layer by liposome fusion, we have investigated these materials, using atomic force microscopy (AFM) and fluorescence microscopy. Force measurements with AFM probes functionalized with bovine serum albumin (BSA) were employed to recognize liposome adsorption. Analysis of the maximal adhesive force and adhesion energy reveals a stronger interaction between BSA and the dextran matrix compared to the lipid-covered surfaces. Images generated using BSA-coated AFM tips indicated a complete and homogeneous coverage of the surface by phospholipid. Single liposomes could not be detected even at lower lipid concentrations, indicating that the liposomes fuse and form a lipid bilayer on the dextran matrix. Experiments with fluorescently labeled liposomes concurred with the AFM studies. Surfaces incubated with liposomes loaded with TRITC-labeled dextran showed no fluorescence, indicating a complete release of the encapsulated dye. In contrast, surfaces incubated with liposomes containing a fluorescently labeled lipid showed fluorescence.     

The effects of liposome size and surface charge on liposome-mediated delivery of methotrexate-γ-aspartate to cells in vitro

We have studied the liposome-mediated delivery of methotrexate-γ-aspartate to five cell lines. The sensitivity of the cells to encapsulated drug varies widely in accordance with their ability to take up the liposomes. CV1-P cells can be 150-times more sensitive to encapsulated methotrexate-γ-aspartate than to free drug, while AKR/J SL2 cells are only twice as sensitive to the encapsulated drug. Negatively-charged liposomes are much more efficient for delivery than are neutral liposomes, and cholesterol is an essential component of the liposome membrane for optimal drug delivery. The optimal liposome size for drug delivery is 0.1 μm, although the amount of cell-associated lipid is the same for all liposome sizes. The effect of the encapsulated drug is inhibited by NH₄Cl, suggesting an endocytic mechanism for delivery. The potency of the encapsulated drug is not affected by wide variations in the drug:lipid ratio.     

AFM characterization of tilt and intrinsic flexibility of Rhodobacter sphaeroides light harvesting complex 2 (LH2)

Atomic force microscopy (AFM) has developed into a powerful tool to investigate membrane protein surfaces in a close-to-native environment. Here we report on the surface topography of Rhodobacter sphaeroides light harvesting complex 2 (LH2) reconstituted into two-dimensional crystals. These photosynthetic trans-membrane proteins formed cylindrical oligomeric complexes, which inserted tilted into the lipid membrane. This peculiar packing of an integral membrane protein allowed us to determine oligomerization and tilt of the LH2 complexes, but also protrusion height and intrinsic flexibility of their individual subunits. Furthermore the surface contouring reliability and limits of the atomic force microscopy could be studied. The two-dimensional crystals examined had sizes of up to 5 microm and, as revealed by a 10 A cryo electron microscopy projection map, p22(1)2(1) crystal symmetry. The unit cell had dimensions of a = b = 150 A and gamma = 90 degrees, and housed four nonameric complexes, two pointing up and two pointing down. AFM topographs of these 2D crystals had a lateral resolution of 10 A. Further, the high vertical resolution of approximately 1 A, allowed the protrusion height of the cylindrical LH2 complexes over the membrane to be determined. This was maximally 13.1 A on one side and 3.8 A on the other. Interestingly, the protrusion height varied across the LH2 complexes, showing the complexes to be inserted with a 6.2 degree tilt with respect to the membrane plane. A detailed analysis of the individual subunits showed the intrinsic flexibility of the membrane protruding peptide stretches to be equal and independent of their protrusion height. Furthermore, our analysis of membrane proteins within this peculiar packing confirmed the high vertical resolution of the atomic force microscopy on biological samples, and led us to conclude that the image acquisition function was equally accurate for contouring protrusions with heights up to approximately 15 A.     

Adhesively-tensed cell membranes: lysis kinetics and atomic force microscopy probing

Membrane tension underlies a range of cell physiological processes. Strong adhesion of the simple red cell is used as a simple model of a spread cell with a finite membrane tension-a state which proves useful for studies of both membrane rupture kinetics and atomic force microscopy (AFM) probing of native structure. In agreement with theories of strong adhesion, the cell takes the form of a spherical cap on a substrate densely coated with poly-L-lysine. The spreading-induced tension, sigma, in the membrane is approximately 1 mN/m, which leads to rupture over many minutes; and sigma is estimated from comparable rupture times in separate micropipette aspiration experiments. Under the sharpened tip of an AFM probe, nano-Newton impingement forces (10-30 nN) are needed to penetrate the tensed erythrocyte membrane, and these forces increase exponentially with tip velocity ( approximately nm/ms). We use the results to clarify how tapping-mode AFM imaging works at high enough tip velocities to avoid rupturing the membrane while progressively compressing it to a approximately 20-nm steric core of lipid and protein. We also demonstrate novel, reproducible AFM imaging of tension-supported membranes in physiological buffer, and we describe a stable, distended network consistent with the spectrin cytoskeleton. Additionally, slow retraction of the AFM tip from the tensed membrane yields tether-extended, multipeak sawtooth patterns of average force approximately 200 pN. In sum we show how adhesive tensioning of the red cell can be used to gain novel insights into native membrane dynamics and structure.     

Mechanics of Biomimetic Liposomes Encapsulating an Actin Shell

Cell-shape changes are insured by a thin, dynamic, cortical layer of cytoskeleton underneath the plasma membrane. How this thin cortical structure impacts the mechanical properties of the whole cell is not fully understood. Here, we study the mechanics of liposomes or giant unilamellar vesicles, when a biomimetic actin cortex is grown at the inner layer of the lipid membrane via actin-nucleation-promoting factors. Using a hydrodynamic tube-pulling technique, we show that tube dynamics is clearly affected by the presence of an actin shell anchored to the lipid bilayer. The same force pulls much shorter tubes in the presence of the actin shell compared to bare membranes. However, in both cases, we observe that the dynamics of tube extrusion has two distinct features characteristic of viscoelastic materials: rapid elastic elongation, followed by a slower elongation phase at a constant rate. We interpret the initial elastic regime by an increase of membrane tension due to the loss of lipids into the tube. Tube length is considerably shorter for cortex liposomes at comparable pulling forces, resulting in a higher spring constant. The presence of the actin shell seems to restrict lipid mobility, as is observed in the corral effect in cells. The viscous regime for bare liposomes corresponds to a leakout of the internal liquid at constant membrane tension. The presence of the actin shell leads to a larger friction coefficient. As the tube is pulled from a patchy surface, membrane tension increases locally, leading to a Marangoni flow of lipids. As a conclusion, the presence of an actin shell is revealed by its action that alters membrane mechanics.     

Membrane fusion by proline-rich Rz1 lipoprotein, the bacteriophage lambda Rz1 gene product.

The fusogenic properties of Rz1, the proline-rich lipoprotein that is the bacteriophage lambda Rz1 gene product, were studied. Light scattering was used to monitor Rz1-induced aggregation of artificial neutral (dipalmitoylphosphatidylcholine/cholesterol) and negatively charged (dipalmitoylphosphatidylcholine/cholesterol/dioleoylphosphatidylserin e) liposomes. Fluorescence assays [the resonance energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl)dihexadecanol-sn-glycero-3-phosphoethanolamine lipid fluorescent probes, as well as fluorescent complex formation between terbium ions and dipicolinic acid encapsulated in two liposome populations and calcein fluorescence] were used to monitor Rz1-induced lipid mixing, contents mixing and leakage of neutral and negatively charged liposomes. The results demonstrated that Rz1 caused adhesion of neutral and negatively charged liposomes with concomitant lipid mixing; membrane distortion, leading to the fusion of liposomes and hence their internal content mixing; and local destruction of the membrane accompanied by leakage of the liposome contents. The use of artificial membranes showed that Rz1 induced the fusion of membranes devoid of any proteins. This might mean that the proline stretch of Rz1 allowed interaction with membrane lipids. It is suggested that Rz1-induced liposome fusion was mediated primarily by the generation of local perturbation in the bilayer lipid membrane and to a lesser extent by electrostatic forces.     

Magnetic tweezers: micromanipulation and force measurement at the molecular level

Cantilevers and optical tweezers are widely used for micromanipulating cells or biomolecules for measuring their mechanical properties. However, they do not allow easy rotary motion and can sometimes damage the handled material. We present here a system of magnetic tweezers that overcomes those drawbacks while retaining most of the previous dynamometers properties. Electromagnets are coupled to a microscope-based particle tracking system through a digital feedback loop. Magnetic beads are first trapped in a potential well of stiffness approximately 10(-7) N/m. Thus, they can be manipulated in three dimensions at a speed of approximately 10 microm/s and rotated along the optical axis at a frequency of 10 Hz. In addition, our apparatus can work as a dynamometer relying on either usual calibration against the viscous drag or complete calibration using Brownian fluctuations. By stretching a DNA molecule between a magnetic particle and a glass surface, we applied and measured vertical forces ranging from 50 fN to 20 pN. Similarly, nearly horizontal forces up to 5 pN were obtained. From those experiments, we conclude that magnetic tweezers represent a low-cost and biocompatible setup that could become a suitable alternative to the other available micromanipulators.     

Sensitive force technique to probe molecular adhesion and structural linkages at biological interfaces.

Adhesion and cytoskeletal structure are intimately related in biological cell function. Even with the vast amount of biological and biochemical data that exist, little is known at the molecular level about physical mechanisms involved in attachments between cells or about consequences of adhesion on the material structure. To expose physical actions at soft biological interfaces, we have combined an ultrasensitive transducer and reflection interference microscopy to image submicroscopic displacements of probe contact with a test surface under minuscule forces. The transducer is a cell-size membrane capsule pressurized by micropipette suction where displacement normal to the membrane under tension is proportional to the applied force. Pressure control of the tension tunes the sensitivity in operation over four orders of magnitude through a range of force from 0.01 pN up to the strength of covalent bonds (approximately 1000 pN)! As the surface probe, a microscopic bead is biochemically glued to the transducer with a densely-bound ligand that is indifferent to the test surface. Movements of the probe under applied force are resolved down to an accuracy of approximately 5 nm from the interference fringe pattern created by light reflected from the bead. With this arrangement, we show that local mechanical compliance of a cell surface can be measured at a displacement resolution set by structural fluctuations. When desired, a second ligand is bound sparsely to the probe for focal adhesion to specific receptors in the test surface. We demonstrate that monitoring fluctuations in probe position at low transducer stiffness enhances detection of molecular adhesion and activation of cytoskeletal structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanical analyses of morphological and topological transformation of liposomes

Liposomes are micro-compartments made of lipid bilayer membranes possessing the characteristics quite similar to those of biological membranes. To form artificial cell-like structures, we made liposomes that contained subunit proteins of cytoskeletons: tubulin or actin. Spherical liposomes were transformed into bipolar or cell-like shapes by mechanical forces generated by the polymerization of encapsulated subunits of microtubules. On the other hand, disk- or dumbbell-shaped liposomes were developed by the polymerization of encapsulated actin. Dynamic processes of morphological transformations of liposomes were visualized by high intensity dark-field light microscopy. Topological changes, such as fusion and division of membrane vesicles, play an essential role in cellular activities. To investigate the mechanism of these processes, we visualized the liposomes undergoing topological transformation in real time. A variety of novel topological transformations were found, including the opening-up of liposomes and the direct expulsion of inner vesicles.     

Entrapment of ovalbumin into liposomes--factors affecting entrapment efficiency, liposome size, and zeta potential

Various amounts of Ovalbumin (OVA) were encapsulated into positively and negatively charged multilamellar liposomes, with the aim to investigate the entrapment efficiency in different buffers and to study their effects on the liposome size and zeta potential. Results showed that the entrapment efficiency of OVA in anionic liposomes was the same in 10 mM Phosphate Buffer (PB) as in Phosphate-Buffered Saline (PBS; PB + 0.15 M NaCl). Also, liposome size was approximately 1200 nm for all anionic liposomes incorporating OVA. The entrapment efficiency of OVA in cationic liposomes was highly dependent on ionic strength. The size of cationic liposomes was approximately 1200 nm in PBS, regardless of protein content, but increased with the amount of the incorporated protein in PB. Aggregation of cationic liposomes in PB was observed when the mass of the protein was 2.5 mg or greater. The zeta potential of anionic liposomes was negative and of cationic liposomes positive in the whole range of protein mass tested. These results show how different compositions of lipid and aqueous phases can be used to vary the entrapment efficiency, liposome size, and zeta potential--the factors that are of great importance for the use of liposomes as drug carriers.     

Force spectroscopy of collagen fibers to investigate their mechanical properties and structural organization

Tendons are composed of collagen and other molecules in a highly organized hierarchical assembly, leading to extraordinary mechanical properties. To probe the cross-links on the lower level of organization, we used a cantilever to pull substructures out of the assembly. Advanced force probe technology, using small cantilevers (length <20 microm), improved the force resolution into the sub-10 pN range. In the force versus extension curves, we found an exponential increase in force and two different periodic rupture events, one with strong bonds (jumps in force of several hundred pN) with a periodicity of 78 nm and one with weak bonds (jumps in force of <7 pN) with a periodicity of 22 nm. We demonstrate a good correlation between the measured mechanical behavior of collagen fibers and their appearance in the micrographs taken with the atomic force microscope.     

Rheological analysis and measurement of neutrophil indentation

Aspects of neutrophil mechanical behavior relevant to the formation of adhesive contacts were assessed by measuring the dependence of the contact area between the cell and a spherical substrate under controlled loading. Micropipettes were used to bring neutrophils into contact with spherical beads under known forces, and the corresponding contact area was measured over time. The neutrophil was modeled as a viscous liquid drop with a constant cortical tension. Both the equilibrium state and the dynamics of the approach to equilibrium were examined. The equilibrium contact area increased monotonically with force in a manner consistent with a cell cortical tension of 16-24 pN/microm. The dynamic response matched predictions based on a model of the cell as a growing drop using published values for the effective viscosity of the cell. The contact pressure between the cell and substrate at equilibrium is predicted to depend on the curvature of the contacting substrate, but to be independent of the impingement force. The approach to equilibrium was rapid, such that the time-averaged stress for a two-second impingement was within 20% of the equilibrium value. These results have implications for the role of mechanical force in the formation of adhesive contacts.     

Entrapment of Ovalbumin into Liposomes—Factors Affecting Entrapment Efficiency,Liposome Size,and Zeta Potential

Various amounts of Ovalbumin (OVA) were encapsulated into positively and negatively charged multilamellar liposomes, with the aim to investigate the entrapment efficiency in different buffers and to study their effects on the liposome size and zeta potential. Results showed that the entrapment efficiency of OVA in anionic liposomes was the same in 10 mM Phosphate Buffer (PB) as in Phosphate-Buffered Saline (PBS; PB?+?0.15 M NaCl). Also, liposome size was approximately 1200 nm for all anionic liposomes incorporating OVA. The entrapment efficiency of OVA in cationic liposomes was highly dependent on ionic strength. The size of cationic liposomes was approximately 1200 nm in PBS, regardless of protein content, but increased with the amount of the incorporated protein in PB. Aggregation of cationic liposomes in PB was observed when the mass of the protein was 2.5 mg or greater. The zeta potential of anionic liposomes was negative and of cationic liposomes positive in the whole range of protein mass tested. These results show how different compositions of lipid and aqueous phases can be used to vary the entrapment efficiency, liposome size, and zeta potential—the factors that are of great importance for the use of liposomes as drug carriers.