Apparent viscosity and cortical tension of blood granulocytes determined by micropipet aspiration.

Continuous deformation and entry flow of single blood granulocytes into small caliber micropipets at various suction pressures have been studied to determine an apparent viscosity for the cell contents and to estimate the extent that dissipation in a cortical layer adjacent to the cell surface contributes to the total viscous flow resistance. Experiments were carried out with a wide range of pipet sizes (2.0-7.5 microns) and suction pressures (10(2)-10(4) dyn/cm2) to examine the details of the entry flow. The results show that the outer cortex of the cell maintains a small persistent tension of approximately 0.035 dyn/cm. The tension creates a threshold pressure below which the cell will not enter the pipet. The superficial plasma membrane of these cells appears to establish an upper limit to surface dilation which is reached after microscopic "ruffles" and "folds" have been pulled smooth. With aspiration of cells by small pipets (less than 2.7 microns), the limit to surface expansion was derived from the maximal extension of the cell into the pipet; final areas were measured to be 2.1 to 2.2 times the area of the initial spherical shape. For suctions in excess of a threshold, the response to constant pressure was continuous flow in proportion to excess pressure above the threshold with only a small nonlinearity over time until the cell completely entered the pipet (for pipet calibers greater than 2.7 microns). With a theoretical model introduced in a companion paper, (Yeung, A., and E. Evans., 1989, Biophys. J. 56:139-149) the entry flow response versus pipet size and suction pressure was analyzed to estimate the apparent viscosity of the cell interior and the ratio of cortical flow resistance to flow resistance from the cell interior. The apparent viscosity was found to depend strongly on temperature with values on the order of 2 x 10(3) poise at 23 degrees C, lower values of 1 x 10(3) poise at 37 degrees C, but extremely large values in excess of 10(4) poise below 10 degrees C. Because of scatter in cell response, it was not possible to accurately establish the characteristic ratio for flow resistance in the cortex to that inside the cell; however, the data showed that the cortex does not contribute significantly to the total flow resistance.     

Extensional flow of erythrocyte membrane from cell body to elastic tether. II. Experiment.

This is the second of two papers on an analytical and experimental study of the flow of erythrocyte membrane. In the experiments discussed here, preswollen human erythrocytes are sphered by aspirating a portion of the cell membrane into a small micropipette; and long, thin, membrane filaments or tethers are steadily withdrawn from the cell at a point diametrically opposite to the point of aspiration. The aspirated portion of the membrane furnishes a reservoir of material that replaces the membrane as it flows as a liquid from the nearly spherical cell body to the cylindrical tether. The application of the principle of conservation of mass permits the tether radius Rt to be measured with the light microscope as the tether is formed and extended at a constant rate. The tether behaves as an elastic solid such that the tether radius decreases as the force or axial tension acting on the tether is increased. For the range of values for Rt is these experiments (100 A less than or equal to Rt less than or equal to 200 A), the slope of the tether-force, tether-radius line is -1.32 dyn/cm. The surface viscosity of the membrane as it flows from cell body to tether is 3 x 10(-3) dyn.s/cm. This viscosity is essentially constant for characteristic rates of deformation between 10 and 200 s-1.     

Temperature dependence of the yield shear resultant and the plastic viscosity coefficient of erythrocyte membrane. Implications about molecular events during membrane failure.

Structural failure of the erythrocyte membrane in shear deformation occurs when the maximum shear resultant (force/length) exceeds a critical value, the yield shear resultant. When the yield shear resultant is exceeded, the membrane flows with a rate of deformation characterized by the plastic viscosity coefficient. The temperature dependence of the yield shear resultant and the plastic viscosity coefficient have been measured over the temperature range 10-40 degrees C. Over this range the yield shear resultant does not change significantly (+/- 15%), but the plastic viscosity coefficient changes exponentially from a value of 1.3 X 10(-2) surface poise (dyn s/cm) at 10 degrees C to a value of 6.2 X 10(-4) surface poise (SP) at 40 degrees C. The different temperature dependence of these two parameters is not surprising, inasmuch as they characterize different molecular events. The yield shear resultant depends on the number and strength of intermolecular connections within the membrane skeleton, whereas the plastic viscosity depends on the frictional interactions between molecular segments as they move past one another in the flowing surface. From the temperature dependence of the plastic viscosity, a temperature-viscosity coefficient, E, can be calculated: eta p = constant X exp(--E/RT). This quantity (E) is related to the probability that a molecular segment can "jump" to its next location in the flowing network. The temperature-viscosity coefficient for erythrocyte membrane above the elastic limit is calculated to be 18 kcal/mol, which is similar to coefficients for other polymeric materials.     

Surface viscosity measurements from large bilayer vesicle tether formation. II. Experiments.

A mechanical experiment has been developed that measures an upper bound for the viscosity of a lipid bilayer membrane. In this experiment, strands of membrane (tethers) are formed from phospholipid vesicles attached to micropipettes by subjecting the vesicles to fluid drag. The rate of tether formation is measured as a function of the velocity of the suspending fluid. The surface viscosity can be calculated from this data using a theoretical relationship derived in a companion paper. Because of the multilamellar character of the vesicles, these values provide an upper bound for the viscosity of a single bilayer. The smallest values obtained in these measurements fell in the range 5.0-13.0 x 10(-6) dyn s/cm. These values are in relatively good agreement with the values calculated from lateral and rotational mobility measurements.     

The viscosity of neutrophils and their transit times through small pores

Passive neutrophils from five different individuals are rapidly aspirated at constant suction pressure and at room temperature into a pipet with a diameter of 4 microns. The excess suction pressures (i.e., the pressures in excess of the small threshold pressure required to produce continuous flow into the pipet) are 5000, 10,000 and 20,000 dyn/cm2 (0.5, 1 and 2 kPa) and are comparable to those encountered in the microcirculation. The rate of entry into the pipet is modeled with a linearized version of a theory by Yeung and Evans for the newtonian flow of a neutrophil into a pipet or pore. From this theory and measurements of the cell size and its rate of entry into the pipet, we can calculate a value for the cytoplasmic viscosity. A linear (newtonian) fit of the theory to the experimental data gives a value for the viscosity of 1050 poise. A non-linear fit predicts a decrease in the "apparent viscosity" from about 1500 poise at zero excess pressure to 1000 poise at an excess aspiration pressure of 20,000 dyn/cm2. Our experiments and analysis also allow us to calculate a value for the transit time through short pores over a wide range of excess aspiration pressures and pore diameters. For example, for a pore diameter of 3 microns and an aspiration pressure of 1250 dyn/cm2, we predict a transit time of about 70 s. At 6 microns and 20,000 dyn/cm2, the predicted transit time is only about 0.04 s.     

Repulsive interactions between uncharged bilayers. Hydration and fluctuation pressures for monoglycerides.

Pressure versus distance relations have been obtained for solid (gel) and neat (liquid-crystalline) phase uncharged lipid bilayers by the use of x-ray diffraction analysis of osmotically stressed monoglyceride aqueous dispersions and multilayers. For solid phase monoelaidin bilayers, the interbilayer repulsive pressure decays exponentially from a bilayer separation of approximately 7 A at an applied pressure of 3 x 10(7) dyn/cm2 to a separation of approximately 11 A at zero applied pressure, where an excess water phase forms. The decay length is approximately 1.3 A, which is similar to the value previously measured for gel phase phosphatidylcholine bilayers. This implies that the decay length of the hydration pressure does not depend critically on the presence of zwitterionic head groups in the bilayer surface. For liquid-crystalline monocaprylin, the repulsive pressure versus distance curve has two distinct regions. In the first region, for bilayer separations of approximately 3-8 A and applied pressures of 3 x 10(8) to 4 x 10(6) dyn/cm2, the pressure decays exponentially with a decay length of approximately 1.3 A. In the second region, for bilayer separations of approximately 8-22 A and applied pressures of 4 x 10(6) to 1 x 10(5) dyn/cm2, the pressure decays much more gradually and is inversely proportional to the cube of the distance between bilayers. These data imply that two repulsive pressures operate between liquid-crystalline monocaprylin bilayers, the hydration pressure, which dominates at small (3-8 A) bilayer separations, and the fluctuation pressure, which dominates at larger bilayer separations (greater than 8 A) and strongly influences the hydration properties of the liquid-crystalline bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elastic area compressibility modulus of red cell membrane.

Micropipette measurements of isotropic tension vs. area expansion in pre-swollen single human red cells gave a value of 288 +/- 50 SD dyn/cm for the elastic, area compressibility modulus of the total membrane at 25 degrees C. This elastic constant, characterizing the resistance to area expansion or compression, is about 4 X 10(4) times greater than the elastic modulus for shear rigidity; therefore, in situations where deformation of the membrane does not require large isotropic tensions (e.g., in passage through normal capillaries), the membrane can be treated by a simple constitutive relation for a two-dimensionally, incompressible material (i.e. fixed area). The tension was found to be linear and reversible for the range of area changes observed (within the experimental system resolution of 10%). The maximum fractional area expansion required to produce lysis was uniformly distributed between 2 and 4% with 3% average and 0.7% SD. By heating the cells to 50 degrees C, it appears that the structural matrix (responsible for the shear rigidity and most of the strength in isotropic tension) is disrupted and primarily the lipid bilayer resists lysis. Therefore, the relative contributions of the structural matrix and lipid bilayer to the elastic, area compressibility could be estimated. The maximum isotropic tension at 25 degrees C is 10-12 dyn/cm and at 50 degrees C is between 3 and 4 dyn/cm. From this data, the respective compressibilities are estimated at 193 dyn/cm and 95 dyn/cm for structural network and bilayer. The latter value correlates well with data on in vitro, monolayer surface pressure versus area curves at oil-water interfaces.     

ETA-receptor blockade impairs vasoconstriction after hemorrhage in xenon-anesthetized dogs treated with an AT1-receptor antagonist

The effects of endothelin receptor subtype A (ETA) blockade on hemodynamics and hormonal adaptation during hemorrhage were studied in xenon/remifentanil-anesthetized dogs (n=6) pretreated with an angiotensin II type 1 (AT1)-receptor blocker. Controls: after a baseline awake period, anesthesia was induced in the dogs with propofol and maintained with xenon/remifentanil (baseline anesthesia). Sixty minutes later, 20 mL x kg(-1) of blood was withdrawn within 5 min and the dogs observed for another hour (hemorrhage). AT1 group followed the same protocol as controls except the AT1-receptor blocker losartan (i.v. 100 microg x kg(-1) x min(-1)) was started at the beginning of the experiment. AT1+ETA group was the same as AT1 group but with the addition of the ETA-receptor blocker atrasentan (i.v. 1 mg x kg(-1), then 0.01 mg x kg(-1) x min(-1)). In controls, mean arterial pressure (MAP) remained unchanged during baseline anesthesia, whereas systemic vascular resistance (SVR) increased from 3282+/-281 to 7321+/-803 dyn.s.cm-5, heart rate (HR) decreased from 86+/-4 to 40+/-3 beats x min(-1), and cardiac output (CO) decreased from 2.3+/-0.2 to 0.9+/-0.1 L x min(-1) (p<0.05), with no further changes after hemorrhage. In AT1-inhibited dogs, MAP (71+/-6 mm Hg) and SVR (5939+/-611 dyn x s x cm(-5)) were lower during baseline anesthesia and after hemorrhage, but greater than those in AT1+ETA (66+/-7 mm Hg, 5034+/-658 dyn x s x cm(-5)) (p<0.05). HR and CO were not different between groups. Plasma concentration of vasopressin was highest with AT1+ETA inhibition after hemorrhage. Combined AT1+ETA-receptor blockade impaired vasoconstriction more than did AT1-receptor blockade alone, both during baseline xenon anesthesia and after hemorrhage. Even a large increase in vasoconstrictor hormones could not prevent the decrease in blood pressure and the smaller increase in SVR. Thus, endothelin is an important vasoconstrictor during hemorrhage, and both endothelin and angiotensin II are essential hormones for cardiovascular stabilization after hemorrhage.     

Viscosity of passive human neutrophils undergoing small deformations.

At issue is the type of constitutive equation that can be used to describe all possible types of deformation of the neutrophil. Here a neutrophil undergoing small deformations is studied by aspirating it into a glass pipet with a diameter that is only slightly smaller than the diameter of the spherically shaped cell. After being held in the pipet for at least seven seconds, the cell is rapidly expelled and allowed to recover its undeformed, spherical shape. The recovery takes approximately 15 s. An analysis of the recovery process that treats the cell as a simple Newtonian liquid drop with a constant cortical (surface) tension gives a value of 3.3 x 10(-5) cm/s for the ratio of the cortical tension to cytoplasmic viscosity. This value is about twice as large as a previously published value obtained with the same model from studies of large deformations of neutrophils. This discrepancy indicates that the cytoplasmic viscosity decreases as the amount of deformation decreases. An extrapolated value for the cytoplasmic viscosity at zero deformation is approximately 600 poise when a value for the cortical tension of 0.024 dyn/cm is assumed. Clearly the neutrophil does not behave like a simple Newtonian liquid drop in that small deformations are inherently different from large deformations. More complex models consisting either of two or more fluids or multiple shells must be developed. The complex structure inside the neutrophil is shown in scanning electron micrographs of osmotically burst cells and cells whose membrane has been dissolved away.     

Micropipette aspiration on the outer hair cell lateral wall

The mechanical properties of the lateral wall of the guinea pig cochlear outer hair cell were studied using the micropipette aspiration technique. A fire-polished micropipette with an inner diameter of approximately 4 microm was brought into contact with the lateral wall and negative pressure was applied. The resulting deformation of the lateral wall was recorded on videotape and subjected to morphometric analysis. The relation between the length of the aspirated portion of the cell and aspiration pressure is characterized by the stiffness parameter, K(s) = 1.07 +/- 0.24 (SD) dyn/cm (n = 14). Values of K(s) do not correlate with the original cell length, which ranges from 29 to 74 microm. Theoretical analysis based on elastic shell theory applied to the experimental data yields an estimate of the effective elastic shear modulus, mu = 15.4 +/- 3.3 dyn/cm. These data were obtained at subcritical aspiration pressures, typically less than 10 cm H2O. After reaching a critical (vesiculation) pressure, the cytoplasmic membrane appeared to separate from the underlying structures, a vesicle with a length of 10-20 microm was formed, and the cytoplasmic membrane resealed. This vesiculation process was repeated until a cell-specific limit was reached and no more vesicles were formed. Over 20 vesicles were formed from the longest cells in the experiment.     

A sensitive measure of surface stress in the resting neutrophil.

The simplest parameterized model of the "passive" or "resting receptive" neutrophil views the cell as being composed of an outer cortex surrounding an essentially liquid-like highly viscous cytoplasm. This cortex has been measured to maintain a small persistent tension of approximately 0.035 dyn/cm (Evans and Yeung. 1989. Biophys. J. 56:151-160) and is responsible for recovering the spherical shape of the cell after large deformation. The origin of the cortical tension is at present unknown, but speculations are that it may be an active process related to the sensitivity of a given cell to external stimulation and the "passive-active" transition. In order to characterize further this feature of the neutrophil we have used a new micropipet manipulation method to give a sensitive measure of the surface stress as a function of the surface area dilation of the highly ruffled cellular membrane. In the experiment, a single cell is driven down a tapered pipet in a series equilibrium deformation positions. Each equilibrium position represents a balance between the stress in the membrane and the pressure drop across the cell. For most cells that seemed to be "passive," as judged by their spherical appearance and lack of pseudopod activity, area dilations of approximately 30% were accompanied by only a small increase in the membrane tension, indicative of a very small apparent elastic area expansion modulus (approximately 0.04 dyn/cm). Extrapolations back to zero area dilation gave a value for the tension in the resting membrane of 0.024 +/- 0.003 dyn/cm, in close agreement with earlier measures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reconstituting the barrier properties of a water-tight epithelial membrane by design of leaflet-specific liposomes

To define aspects of lipid composition and bilayer asymmetry critical to barrier function, we examined the permeabilities of liposomes that model individual leaflets of the apical membrane of a barrier epithelium, Madin-Darby canine kidney type 1 cells. Using published lipid compositions we prepared exofacial liposomes containing phosphatidylcholine, sphingomyelin, glycosphingolipids, and cholesterol; and cytoplasmic liposomes containing phosphatidylethanolamine, phosphatidylserine, and cholesterol. The osmotic permeability of cytoplasmic liposomes to water (P(f)), solutes, and NH(3) was 18-90-fold higher than for the exofacial liposomes (P(f(ex)) = 2.4 +/- 0.4 x 10(-4) cm/s, P(f(cy)) = 4.4 +/- 0.3 x 10(-3) cm/s; P(glycerol(ex)) = 2.5 +/- 0.3 x 10(-8) cm/s, P(glycerol(cy)) = 2.2 +/- 0.02 x 10(-6) cm/s; P(NH3(ex)) = 0. 13 +/- 0.4 x 10(-4) cm/s, P(NH3(cy)) = 7.9 +/- 1.0 x 10(-3) cm/s). By contrast, the apparent proton permeability of exofacial liposomes was 4-fold higher than cytoplasmic liposomes (P(H+(ex)) = 1.1 +/- 0. 1 x 10(-2) cm/s, P(H+(cy)) = 2.7 +/- 0.6 x 10(-3) cm/s). By adding single leaflet permeabilities, we calculated a theoretical P(f) for a Madin-Darby canine kidney apical membrane of 4.6 x 10(-4) cm/s, which compares favorably with experimentally determined values. In exofacial liposomes lacking glycosphingolipids or sphingomyelin, permeabilities were 2-7-fold higher, indicating that both species play a role in barrier function. Removal of cholesterol resulted in 40-280-fold increases in permeability. We conclude: 1) that we have reconstituted the biophysical properties of a barrier membrane, 2) that the barrier resides in the exofacial leaflet, 3) that both sphingomyelin and glycosphingolipids play a role in reducing membrane permeability but that there is an absolute requirement for cholesterol to mediate this effect, 4) that these results further validate the hypothesis that each leaflet offers an independent resistance to permeation, and 5) that proton permeation was enhanced by sphingolipid/cholesterol interactions.     

Anacrotic "notch" on the upstroke of external carotid curve may indicate a critically high systolic pulmonary arterial pressure value.

Acute respiratory failure is followed by decreased left ventricular performance probably due to the right ventricle dilatation induced by pulmonary hypertension and intraventricular septal shift to the left. An anacrotic notch on the upstroke slope of the carotid curve was detected in 22 of 36 hemodynamic studies with simultaneous ECG, PCG and external pulse carotid curve recording in 7 burned patients with acute respiratory failure. Comparing the values (x +/- SEM) obtained in group with notch and in group without notch, PAPs, PAPm, PVRI were higher (56 +/- 2.30 mmHg; 32 +/- 0.99 mm Hg; 543 +/- 56.8 dyn x s/cm5/m2 versus 32 +/- 1.08 mm Hg; 20 +/- 0.9 mm Hg; 173 +/- 14.7 dyn x s/cm5/m2) and CI and LVSWI were lower (2.6 +/- 0.17 l/min/m2; 25.8 +/- 2.41 g x m/m2; versus 3.8 +/- 0.26 l/min/m2; 38.3 +/- 2.82 g x m/m2) in group with notch. As it is shown by 11 paired measurements where the notch disappeared immediately after starting vasodilator therapy PAPs, PAPm, PVRI decreased (from 54 +/- 3.1, 35 +/- 0.8 mm Hg, 498 +/- 64.1 dyn x s/cm5/m2 to 35 +/- 0.8, 21 +/- 1.1 mmHg, 189 +/- 18.4 dyn x s/cm5/m2 respectively) and heart performance improved. Since the left ventricle contractility (characterized by EF, PCWP, ICT) was normal in both groups, our findings suggest that critically high PAPs values (over 40 mmHg) cause a septal bulging at the beginning of the systole which in turn narrows the left ventricle outflow tract. Regarding to the clinical importance of the deteriorated biventricular function at the critically high PAPs evidenced by notch phenomenon on carotid curve but measurable only by indwelling pulmonary arterial catheterization always being a source of infection, the noninvasive parameters as independent variables were entered into canonical discriminant analysis. The ratio of the correctly classified cases was 89%.     

Integrated L-phenylalanine separation in an E. coli fed-batch process: from laboratory to pilot scale

Pilot-scale reactive-extraction technology for fully integrated L-phenylalanine (L-Phe) separation in Escherichia coli fed-batch fermentations was investigated in order to prevent an inhibition of microbial L-Phe production by-product accumulation. An optimal reactive-extraction system, consisting of an organic kerosene phase with the cation-selective carrier DEHPA (di-2-ethylhexyl phosphonic acid) and an aqueous stripping phase including sulphuric acid, was found particularly efficient. Using this system with two membrane contactors, mass-transfer coefficients of up to 288 x 10(-7) cm s(-1) for the aqueous/organic and 77 x 10(-7) cm s(-1) for the organic/stripping phase were derived from experimental data using a simple modelling approach. Concentration factors higher than 4 were achieved in the stripping phase as compared to the aqueous donor phase. Reactive extraction enabled a 98% cation portion of L-Phe in the stripping phase, leading to final product purity higher than 99% after L-Phe precipitation. A doubling of L-Phe/glucose yield was observed when kerosene/DEHPA was added to the fermentation solution in the bioreactor to experimentally simulate a fully integrated L-Phe separation process.     

Elasticity of synthetic phospholipid vesicles and submitochondrial particles during osmotic swelling

A rapid and accurate method has been developed for measuring the elastic response of vesicle bilayer membranes to an applied osmotic pressure. The technique of dynamic light scattering is used to measure both the elastic constant and the elastic limit of dioleoylphosphatidic acid (DOPA) and DOPA-cholesterol vesicles and of submitochondrial particles derived from the inner membrane of bovine heart mitochondria. The vesicles prepared by the pH-adjustment method are unilamellar and of uniform size between 240 and 460 nm in diameter. The vesicles swell uniformly upon dilution. The observed change in size is not due to any change in the shape of the vesicles. The data also indicate that the vesicles are spherical and not flaccid. The total vesicle swelling in these studies resulted in a 3-4% increase in surface area for vesicles swollen in 0.15 M KCl and a 5-10% increase in surface area for vesicles swollen in 0.25 M sucrose. This maximum represents the elastic limit of the vesicles. Evidence is presented to show that the vesicles release contents after swelling to this maximum, reseal immediately, and reswell according to the osmotic pressure. For DOPA vesicles in a 0.15 M KCl-tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer (pH 7.55), the observed membrane modulus is found to be in the range of 10(8) dyn/cm2. The modulus was found to be in the order of 10(7) dyn/cm2 for DOPA vesicles in a 0.25 M sucrose-Tris-HCl buffer (pH 7.55). This is comparable to that of submitochondrial particles in the same sucrose-Tris-HCl buffer. The observed membrane modulus also decreases with vesicle size. Its magnitude and its variation with ionic strength indicate that the major component of bilayer elasticity is neither the inherent elasticity of the bilayer nor the bending modulus. The variation of the membrane modulus with respect to curvature suggests that its principal component may be related to surface tension effects including the negative charges on the vesicle surface. There is considerable variation between vesicles swollen in sucrose and those swollen in KCl in the membrane modulus, in the elastic limit at which the vesicles burst, and in the transbilayer pressure difference at bursting. The latter was found to be 4-6 mosM (10(5) dyn/cm2) in sucrose solution and 20-4 mosM (10(6) dyn/cm2) in KCl solution.     

Membrane viscoplastic flow.

In this paper, a theory of viscoplasticity formulated by Prager and Hohenemser is developed for a two-dimensional membrane surface and applied to the analysis of the flow of "microtethers" pulled from red blood cells attached to glass substrates. The viscoplastic flow involves two intrinsic material constants: yield shear and surface viscosity. The intrinsic viscosity for plastic flow of membrane is calculated to be 1 X 10(-2) dyn-s/cm from microtether flow experiments, three orders of magnitude greater than surface viscosities of lipid membrane components. The fluid dissipation is dominated by the flow of a structural matrix which has exceeded its yield shear. The yield shear is the maximum shear resultant that the membrane can sustain before it begins to deform irreversibly. The yield shear is found to be in the range 2-8 X 10(-2) dyn/cm, two or three orders of magnitude smaller than the isotropic tension required to lyse red cells.     

Adiabatic compressibility of myosin subfragment-1 and heavy meromyosin with or without nucleotide.

The partial specific adiabatic compressibilities of myosin subfragment-1 (S1) and heavy meromyosin (HMM) of skeletal muscle in solution were determined by measuring the density and the sound velocity of the solution. The partial specific volumes of S1 and HMM were 0.713 and 0.711 cm3/g, respectively. The partial specific adiabatic compressibilities of S1 and HMM were 4.2 x 10(-12) and 2.9 x 10(-12) cm2/dyn, respectively. These values are in the same range as the most of globular proteins so far studied. The result indicates that the flexibility of S1 region almost equals to that of HMM. After binding to ADP.orthovanadate, S1 and HMM became softer than their complexes with ADP. The bulk moduli of S1 and HMM were of the order of (4-6) x 10(10) dyn/cm2, which are very comparable with the bulk modulus of muscle fiber.     

Thermoelasticity of large lecithin bilayer vesicles.

Micromechanical experiments on large lecithin bilayer vesicles as a function of temperature have demonstrated an essential feature of bilayer vesicles as closed systems: the bilayer can exist in a tension-free state (within the limits of experimental resolution, i.e., less than 10(-2) dyn/cm). Furthermore, because of the fixed internal volume, there is a critical temperature at which the vesicle becomes a tension-free sphere. Below this temperature, thermoelastic tension builds up in the membrane and the vesicle's internal pressure increases while the surface area remains constant. Above this temperature, the vesicle's surface area increases while the tension and internal pressure are negligible. Without mechanical support, the vesicles fragment into small vesicles because they have insufficient surface rigidity. In the upper temperature range we have measured the increase of surface area with temperature. These data established the thermal area expansivity to be 2.4 X 10(-3)/degrees C. At constant temperature, we used either pipet aspiration with suction pressures up to 10(4) dyn/cm2 or compression against a flat surface with forces up to 10(-2) dyn to produce area dilation of the vesicle surface on the order of 1%. The rate of increase of membrane tension with area dilation was calculated, which established the elastic area compressibility modulus to be 140 dyn/cm. The tension limit that produced lysis was observed to be 3-4 dyn/cm (equivalent to 2-3% area increase). The product of the elastic area compressibility modulus, the thermal area expansivity, and the temperature gives the reversible heat of expansion at constant temperature for the bilayer. This value is 100 ergs/cm2 at 25 degrees C, or approximately 5 kcal/mol of lecithin. Similarly, the product of the thermal area expansivity multiplied by the area compressibility modulus determines the rate of increase of thermoelastic tension with decrease in temperature when the area is held constant, i.e., -0.34 dyn/cm/degrees C.     

Effect of cell substratum on lateral mobility of lipids in the plasma membrane of vascular endothelial cells

Bovine vascular endothelial cells can be maintained in a highly differentiated state in vitro, either by the addition of fibroblast growth factor (FGF) to the culture medium or by plating the cells on extracellular matrix (ECM)-coated dishes. Under these conditions the cells proliferate actively and at confluence form a tightly packed monolayer composed of nonoverlapping polarized cells. A fluorescence recovery after photobleaching method was used to determine the lateral mobility coefficient D of the lipophilic fluorescent probe, 5N-(hexadecanoyl)-aminofluorescein (HEDAF), in the basal and apical plasma membranes of endothelial cells under various culture conditions (cells on glass coverslips in the presence or absence of FGF, or cells plated on ECM in the exponential growth phase or at confluence). A heterogeneous distribution of lateral diffusion coefficients D was found in a given cell population. Nevertheless, for the basal membrane, a "mean" D value close to 2.0 x 10(-9) cm2/s was found for all the culture conditions. The "mean" D value of HEDAF in the apical pole was slightly higher when sparse cells were exposed to FGF (D = 2.2 x 10(-9) cm2/s) and was further enhanced when cells were growing or confluent on ECM-coated coverslips (D = 2.7 x 10(-9) cm2/s). On the other hand, when the cells were maintained in the absence of FGF on glass coverslips, similar "mean" D values were found in both cell poles (D = 2.0 x 10(-9) cm2/s). These results show that lateral mobility of lipids in endothelial plasmalemma varies in response to external factors such as FGF and the ECM.     

Unconfined lateral diffusion and an estimate of pericellular matrix viscosity revealed by measuring the mobility of gold-tagged lipids

Nanovid (video-enhanced) microscopy was used to determine whether lateral diffusion in the plasma membrane of colloidal gold-tagged lipid molecules is confined or is unrestricted. Confinement could be produced by domains within the plane of the plasma membrane or by filamentous barriers within the pericellular matrix. Fluorescein- phosphatidylethanolamine (F1-PE), incorporated into the plasma membranes of cultured fibroblasts, epithelial cells and keratocytes, was labeled with 30-nm colloidal gold conjugated to anti-fluorescein (anti-F1). The trajectories of the gold-labeled lipids were used to compute diffusion coefficients (DG) and to test for restricted motion. On the cell lamella, the gold-labeled lipids diffused freely in the plasma membrane. Since the gold must move through the pericellular matrix as the attached lipid diffuses in the plasma membrane, this result suggests that any extensive filamentous barriers in the pericellular matrix are at least 40 nm from the plasma membrane surface. The average diffusion coefficients ranged from 1.1 to 1.7 x 10(-9) cm2/s. These values were lower than the average diffusion coefficients (DF) (5.4 to 9.5 x 10(-9) cm2/s) obtained by FRAP. The lower DG is partially due to the pericellular matrix as demonstrated by the result that heparinase treatment of keratocytes significantly increased DG to 2.8 x 10(-9) cm2/s, but did not affect DF. Pericellular matrix viscosity was estimated from the frictional coefficients computed from DG and DF and ranged from 0.5 to 0.9 poise for untreated cells. Heparinase treatment of keratocytes decreased the apparent viscosity to approximately 0.1 poise. To evaluate the presence of domains or barriers, the trajectories and corresponding mean square displacement (MSD) plots of gold-labeled lipids were compared to the trajectories and MSD plots resulting from computer simulations of random walks within corrals. Based on these comparisons, we conclude that, if there are domains limiting the diffusion of F1-PE, most are larger than 5 microns in diameter.