Passive mechanical behavior of human neutrophils: effect of cytochalasin B.

Actin is a ubiquitous protein in eukaryotic cells. It plays a major role in cell motility and in the maintenance and control of cell shape. In this article, we intend to address the contribution of actin to the passive mechanical properties of human neutrophils. As a framework for assessing this contribution, the neutrophil is modeled as a simple viscous fluid drop with a constant cortical ("surface") tension. The reagent cytochalasin B (CTB) was used to disrupt the F-actin structure, and the neutrophil cortical tension and cytoplasmic viscosity were evaluated by single-cell micropipette aspiration. The cortical tension was calculated by simple force balance, and the viscosity was calculated according to a numerical analysis of the cell entry into the micropipette. CTB reduced the cell cortical tension in a dose-dependent fashion: by 19% at a concentration of 3 microM and by 49% at 30 microM. CTB also reduced the cytoplasmic viscosity by approximately -25% at a concentration of 3 microM and by approximately 65% at a concentration of 30 microM when compared at the same aspiration pressures. All three groups of neutrophils, normal cells, and cells treated with either 3 or 30 microM CTB, exhibited non-Newtonian behavior, in that the apparent viscosity decreased with increasing shear rate. The dependence of the cytoplasmic viscosity on deformation rate can be described empirically by mu = mu c(gamma m/gamma c)-b, where mu is cytoplasmic viscosity, gamma m is mean shear rate, mu c is the characteristic viscosity at the characteristic shear rate gamma c, and b is a material coefficient.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Cortical shell-liquid core model for passive flow of liquid-like spherical cells into micropipets.

Many nonadherent cells exist as spheres in suspension and when sucked into pipets, deform continuously like liquids within the fixed surface area limitation of a plasma membrane envelope. After release, these cells eventually recover their spherical form. Consequently, pipet aspiration test provides a useful method to assay the apparent viscosity of such cells. For this purpose, we have analyzed the inertialess flow of a liquid-like model cell into a tube at constant suction pressure. The cell is modeled as a uniform liquid core encapsulated by a distinct cortical shell. The method of analysis employs a variational approach that minimizes errors in boundary conditions defined by the equations of motion for the cortical shell where the trial functions are exact solutions for the flow field inside the liquid core. For the particular case of an anisotropic liquid cortex with persistent tension, we have determined universal predictions for flow rate scaled by the ratio of excess pressure (above the threshold established by the cortical tension) and core viscosity which is the reciprocal of the dynamic resistance to entry. The results depend on pipet to cell size ratio and a parameter that characterizes the ratio of viscous flow resistance in the cortex to that inside the cytoplasmic core. The rate of entry increases markedly as the pipet size approaches the outer segment diameter of the cell. Viscous dissipation in the cortex strongly influences the entry flow resistance for small tube sizes but has little effect for large tubes. This indicates that with sufficient experimental resolution, measurement of cell entry flow with different-size pipets could establish both the cortex to cell dissipation ratio as well as the apparent viscosity of the cytoplasmic core.     

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.     

RNA interference in J774 macrophages reveals a role for coronin 1 in mycobacterial trafficking but not in actin-dependent processes

Macrophages are crucial for innate immunity, apoptosis, and tissue remodeling, processes that rely on the capacity of macrophages to internalize and process cargo through phagocytosis. Coronin 1, a member of the WD repeat protein family of coronins specifically expressed in leukocytes, was originally identified as a molecule that is recruited to mycobacterial phagosomes and prevents the delivery of mycobacteria to lysosomes, allowing these to survive within phagosomes. However, a role for coronin 1 in mycobacterial pathogenesis has been disputed in favor for its role in mediating phagocytosis and cell motility. In this study, a role for coronin 1 in actin-mediated cellular processes was addressed using RNA interference in the murine macrophage cell line J774. It is shown that the absence of coronin 1 in J774 macrophages expressing small interfering RNA constructs specific for coronin 1 does not affect phagocytosis, macropinocytosis, cell locomotion, or regulation of NADPH oxidase activity. However, in coronin 1-negative J774 cells, internalized mycobacteria were rapidly transferred to lysosomes and killed. Therefore, these results show that in J774 cells coronin 1 has a specific role in modulating phagosome-lysosome transport upon mycobacterial infection and that it is dispensable for most F-actin-mediated cytoskeletal rearrangements.     

Passive mechanical behavior of human neutrophils: power-law fluid.

The mechanical behavior of the neutrophil plays an important role in both the microcirculation and the immune system. Several laboratories in the past have developed mechanical models to describe different aspects of neutrophil deformability. In this study, the passive mechanical properties of normal human neutrophils have been further characterized. The cellular mechanical properties were assessed by single cell micropipette aspiration at fixed aspiration pressures. A numerical simulation was developed to interpret the experiments in terms of cell mechanical properties based on the Newtonian liquid drop model (Yeung and Evans, Biophys. J., 56: 139-149, 1989). The cytoplasmic viscosity was determined as a function of the ratio of the initial cell size to the pipette radius, the cortical tension, aspiration pressure, and the whole cell aspiration time. The cortical tension of passive neutrophils was measured to be about 2.7 x 10(-5) N/m. The apparent viscosity of neutrophil cytoplasm was found to depend on aspiration pressure, and ranged from approximately 500 Pa.s at an aspiration pressure of 98 Pa (1.0 cm H2O) to approximately 50 Pa.s at 882 Pa (9.0 cm H2O) when tested with a 4.0-micron pipette. These data provide the first documentation that the neutrophil cytoplasm exhibits non-Newtonian behavior. To further characterize the non-Newtonian behavior of human neutrophils, a mean shear rate gamma m was estimated based on the numerical simulation. The apparent cytoplasmic viscosity appears to decrease as the mean shear rate increases. The dependence of cytoplasmic viscosity on the mean shear rate can be approximated as a power-law relationship described by mu = mu c(gamma m/gamma c)-b, where mu is the cytoplasmic viscosity, gamma m is the mean shear rate, mu c is the characteristic viscosity at characteristic shear rate gamma c, and b is a material coefficient. When gamma c was set to 1 s-1, the material coefficients for passive neutrophils were determined to be mu c = 130 +/- 23 Pa.s and b = 0.52 +/- 0.09 for normal neutrophils. The power-law approximation has a remarkable ability to reconcile discrepancies among published values of the cytoplasmic viscosity measured using different techniques, even though these values differ by nearly two orders of magnitude. Thus, the power-law fluid model is a promising candidate for describing the passive mechanical behavior of human neutrophils in large deformation. It can also account for some discrepancies between cellular behavior in single-cell micromechanical experiments and predictions based on the assumption that the cytoplasm is a simple Newtonian fluid.     

Role of the membrane cortex in neutrophil deformation in small pipets.

The simplest model for a neutrophil in its "passive" state views the cell as consisting of a liquid-like cytoplasmic region surrounded by a membrane. The cell surface is in a state of isotropic contraction, which causes the cell to assume a spherical shape. This contraction is characterized by the cortical tension. The cortical tension shows a weak area dilation dependence, and it determines the elastic properties of the cell for small curvature deformations. At high curvature deformations in small pipets (with internal radii less than 1 micron), the measured critical suction pressure for cell flow into the pipet is larger than its estimate from the law of Laplace. A model is proposed where the region consisting of the cytoplasm membrane and the underlying cortex (having a finite thickness) is introduced at the cell surface. The mechanical properties of this region are characterized by the apparent cortical tension (defined as a free contraction energy per unit area) and the apparent bending modulus (introduced as a bending free energy per unit area) of its middle plane. The model predicts that for small curvature deformations (in pipets having radii larger than 1.2 microns) the role of the cortical thickness and the resistance for bending of the membrane-cortex complex is negligible. For high curvature deformations, they lead to elevated suction pressures above the values predicted from the law of Laplace. The existence of elevated suction pressures for pipets with radii from 1 micron down to 0.24 micron is found experimentally. The measured excess suction pressures cannot be explained only by the modified law of Laplace (for a cortex with finite thickness and negligible bending resistance), because it predicts unacceptable high cortical thicknesses (from 0.3 to 0.7 micron). It is concluded that the membrane-cortex complex has an apparent bending modulus from 1 x 10(-18) to 2 x 10(-18) J for a cortex with a thickness from 0.1 micron down to values much smaller than the radius of the smallest pipet (0.24 micron) used in this study.     

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)     

Stage-specific mRNAs coding for subtypes of H2A and H2B histones in the sea urchin embryo

Phagocytosis, pinocytosis and the surface distribution of concanavalin A (ConA) have been analyzed during mitosis in several mammalian cell lines. Use of the bisbenzimidazole dye, Hoechst 33258, for chromosome staining after gentle fixation made possible the rapid identification and correlation of mitotic phase with surface properties.Phagocytosis of both opsonized and nonopsonized particles is markedly depressed in mitotic cells of the mouse macrophage cell line J774.1. The uptake of opsonized particles (IgG-coated erythrocytes) is Impaired from early prophase through early G1, whereas phagocytosis of non-opsonized particles (latex beads) is restored by telophase. Fluid pinocytosis, determined by the uptake of soluble horseradish peroxidase, is also inhibited during mitosis. Thus peroxidase-containing cytoplasmic vesicles were virtually absent from mid-prophase through telophase in both J774 and Chinese hamster ovary (CHO) cells.Adsorptive pinocytosis of ConA was determined from the different distributions of fluorescence in single cells incubated at 37°C with rhodamine-conjugated ConA (surface and cytoplasmic label), then fixed and further incubated with fluorescein-conjugated anti-ConA (surface only). The separate fluorescence of Hoechst, fluorescein and rhodamine could be optically isolated. In interphase J774 cells, ConA is rapidly internalized into cytoplasmic vesicles. In contrast, ConA is restricted to the plasma membrane from mid-prophase through telophase. In CHO, the depressed pattern of internalization is not fully established until metaphase.The surface distribution of ConA also varied dramatically as a function of mitotic phase. Between mid-prophase and early anaphase, the pattern of surface ConA-receptor complexes is diffuse. Once the cleavage furrow begins to develop, however, ConA moves into the region of the furrow. This was shown in J774, CHO and 3T3 mouse embryonic fibroblasts, and is probably universal. ConA movement into the membrane that overlies the microfilaments of the contractile ring is analogous to similar movements that occur in interphase cells during ConA cap formation and during the development of phagocytic pseudopods. The analogy emphasizes the common functional consequences of microfilament-membrane organization.It is evident that membrane processes which depend upon endocytosis-for example, certain hormone-induced signals-may be interrupted during mitosis. Inhibition of endocytosis thus may be a significant element in the control of cellular activities during mitosis and a strong influence on the properties of the emergent post-mitotic cell.     

Leukocyte rolling on P-selectin: a three-dimensional numerical study of the effect of cytoplasmic viscosity

Rolling leukocytes deform and show a large area of contact with endothelium under physiological flow conditions. We studied the effect of cytoplasmic viscosity on leukocyte rolling using our three-dimensional numerical algorithm that treats leukocyte as a compound droplet in which the core phase (nucleus) and the shell phase (cytoplasm) are viscoelastic fluids. The algorithm includes the mechanical properties of the cell cortex by cortical tension and considers leukocyte microvilli that deform viscoelastically and form viscous tethers at supercritical force. Stochastic binding kinetics describes binding of adhesion molecules. The leukocyte cytoplasmic viscosity plays a critical role in leukocyte rolling on an adhesive substrate. High-viscosity cells are characterized by high mean rolling velocities, increased temporal fluctuations in the instantaneous velocity, and a high probability for detachment from the substrate. A decrease in the rolling velocity, drag, and torque with the formation of a large, flat contact area in low-viscosity cells leads to a dramatic decrease in the bond force and stable rolling. Using values of viscosity consistent with step aspiration studies of human neutrophils (5-30 Pa·s), our computational model predicts the velocities and shape changes of rolling leukocytes as observed in vitro and in vivo.     

Effect of phagocytosis on receptor distribution and endocytic activity in macrophages

Phagocytosis requires the internalization of a significant fraction of the plasma membrane and results in the intracellular deposition of large particles. We evaluated the effect of phagocytosis on the cellular distribution of recycling receptors and uptake of ligand to determine whether phagocytosis affects receptor behavior. Phagocytosis of zymosan, latex particles, or IgG-coated red blood cells by rabbit alveolar macrophages did not decrease the number of cell surface receptors for transferrin, alpha 2-macroglobulin X protease complexes, maleylated proteins, or mannosylated proteins. The number of surface receptors for transferrin was also unaltered in J774 cells, a macrophage-like cell line. In both cell types extensive phagocytosis did not affect the rate of receptor-mediated endocytosis or the distribution of receptors between the endosome and the cell surface. However, fluid phase pinocytosis was reduced by phagocytosis. The major reduction appeared to be not in the rate of internalization but rather in the delivery of fluid to the lysosome. These results demonstrate that internalization of a significant amount of the plasma membrane during phagocytosis does not diminish the number of receptors on the cell surface and has no effect on receptor-mediated ligand uptake.     

Estimating intercellular surface tension by laser-induced cell fusion

Intercellular surface tension is a key variable in understanding cellular mechanics. However, conventional methods are not well suited for measuring the absolute magnitude of intercellular surface tension because these methods require determination of the effective viscosity of the whole cell, a quantity that is difficult to measure. In this study, we present a novel method for estimating the intercellular surface tension at single-cell resolution. This method exploits the cytoplasmic flow that accompanies laser-induced cell fusion when the pressure difference between cells is large. Because the cytoplasmic viscosity can be measured using well-established technology, this method can be used to estimate the absolute magnitudes of tension. We applied this method to two-cell-stage embryos of the nematode Caenorhabditis elegans and estimated the intercellular surface tension to be in the 30-90 μN m(-1) range. Our estimate was in close agreement with cell-medium surface tensions measured at single-cell resolution.     

Leishmania donovani lipophosphoglycan causes periphagosomal actin accumulation: correlation with impaired translocation of PKCα and defective phagosome maturation

Lipophosphoglycan (LPG) is the major surface glycoconjugate of Leishmania donovani promastigotes. The repeating disaccharide–phosphate units of LPG are crucial for promastigote survival inside macrophages and establishment of infection. LPG has a number of effects on the host cell, including inhibition of PKC activity, inhibition of nitric oxide production and altered expression of cytokines. LPG also inhibits phagosomal maturation, a process requiring depolymerization of periphagosomal F-actin. In the present study, we have characterized the dynamics of F-actin during the phagocytosis of L. donovani promastigotes in J774 macrophages. We observed that F-actin accumulated progressively around phagosomes containing wild-type L. donovani promastigotes during the first hour of phagocytosis. Using LPG-defective mutants and yeast particles coated with purified LPG, we obtained evidence that this effect could be attributed to the repeating units of LPG. LPG also disturbed cortical actin turnover during phagocytosis. The LPG-dependent accumulation of periphagosomal F-actin correlated with an impaired recruitment of the lysosomal marker LAMP1 and PKCα to the phagosome. Accumulation of periphagosomal F-actin during phagocytosis of L. donovani promastigotes may contribute to the inhibition of phagosomal maturation by physically preventing vesicular trafficking to and from the phagosome.     

Mechanics of spreading cells probed by atomic force microscopy

Cellular adhesion and motility are fundamental processes in biological systems such as morphogenesis and tissue homeostasis. During these processes, cells heavily rely on the ability to deform and supply plasma membrane from pre-existing membrane reservoirs, allowing the cell to cope with substantial morphological changes. While morphological changes during single cell adhesion and spreading are well characterized, the accompanying alterations in cellular mechanics are scarcely addressed. Using the atomic force microscope, we measured changes in cortical and plasma membrane mechanics during the transition from early adhesion to a fully spread cell. During the initial adhesion step, we found that tremendous changes occur in cortical and membrane tension as well as in membrane area. Monitoring the spreading progress by means of force measurements over 2.5 h reveals that cortical and membrane tension become constant at the expense of excess membrane area. This was confirmed by fluorescence microscopy, which shows a rougher plasma membrane of cells in suspension compared with spread ones, allowing the cell to draw excess membrane from reservoirs such as invaginations or protrusions while attaching to the substrate and forming a first contact zone. Concretely, we found that cell spreading is initiated by a transient drop in tension, which is compensated by a decrease in excess area. Finally, all mechanical parameters become almost constant although morphological changes continue. Our study shows how a single cell responds to alterations in membrane tension by adjusting its overall membrane area. Interference with cytoskeletal integrity, membrane tension and excess surface area by administration of corresponding small molecular inhibitors leads to perturbations of the spreading process.     

A physical characterization of GAP A3 hybridoma cells: morphology, geometry, and mechanical properties

Morphological, geometrical, and rheological properties of the GAP A3 hybridoma cell line have been evaluated as a function of the cell cycle. Interference contrast video microscopy and scanning electron microscopy (SEM) showed that a sample of cells taken from the middle of the exponential growht phase displayed a range of cell morphologies, consistent with a heterogeneous growing culture. Micropipet manipulation was used to measure the geometrical (cell volume) and mechanical (cortical tension and apparent cell viscosity) properties of single cells selected at random from a sample in the middle of the exponential growth phase. Consistent with the range of morphologies, cell volumes (1400 to 5700 mum(3)) and apparent viscosities (430 to 1.2 x 10(4) P) showed a wide range of values at 37 degrees C, demonstrating that a hybridoma cell line cannot be characterized by a single value for any one property, and that properties must be related to their cycle dependence when considering proliferating cells. Direct, video-microscopic observation of synchronized cells, and of individual cells that were followed throughout their cell cycle, allowed us to correlated distinct morphologies with phases of the cell cycle. As the cell cycle progresses, an increase in cell volume by a factor of 3 to 4is accompanied by an overall increase in apparent cell viscosity by approximately the same ratio, consistent with an accumulation of more cytoplasmic material in the older cells. Also, a decrease in average apparent viscosity by a factor of 10. These results are important in order to evaluate the possible role of certain structural, cell-cycle dependent features in shear and abrasion sensitivity. This is a problem of current concern in the bioreactor culture of mammalian cells.     

Predicting the kinetics of cell spreading

We apply the wetting theory to predict the kinetics of fibroblast spreading onto an adhesive substrate, under simplifying assumptions on the cell structure and geometry. Three main parameters are used: cytoplasmic viscosity, cortical tension, and cell-to-substrate adhesion energy. The viscosity and tension values are taken from previous micromechanical studies. The adhesion energy, ill known, is adjusted by fitting the model predictions to available experimental data of contact radius versus time. The agreement is quite good, justifying such a "macroscopic" view of cell morphology.     

Use of cell contour analysis to evaluate the affinity between macrophages and glutaraldehyde-treated erythrocytes.

Recently, several authors evaluated the affinity between lipid bilayers or erythrocyte membranes by analyzing the deformation of cells or vesicles they brought into close contact using micromanipulators. In the present report, we extend this approach in a study of the adhesive properties of rough nucleated cells. Rat peritoneal macrophages were made to bind human red cells modified with glutaraldehyde or glutaraldehyde and polylysine. Conjugates were examined with electron microscopy, and photomicrographs were digitized for quantification of cell surface roughness in and out of adhesion areas. Also, macrophages were subjected to micropipette aspiration to find a relationship between apparent surface tension and area increase. Assuming that this increase was a direct consequence of a smoothing of the cell surface on the submicrometer scale, the actual affinity between macrophages and erythrocytes was estimated. The obtained values ranged between 8.4 X 10(-5) and 18.2 X 10(-5) J/m2. It is concluded that cell surface roughness may be an important parameter of cell adhesion and perhaps deformation. This is made amenable to experimental study by the present approach.     

Cell surface topography controls phagocytosis and cell spreading: The membrane reservoir in neutrophils

Neutrophils exhibit rapid cell spreading and phagocytosis, both requiring a large apparent increase in the cell surface area. The wrinkled surface topography of these cells may provide the membrane reservoir for this. Here, the effects of manipulation of the neutrophil cell surface topography on phagocytosis and cell spreading were established. Chemical expansion of the plasma membrane or osmotic swelling had no effects. However, osmotic shrinking of neutrophils inhibited both cell spreading and phagocytosis. Triggering a Ca²⁺ signal in osmotically shrunk cells (by IP₃ uncaging) evoked tubular blebs instead of full cell spreading. Phagocytosis was halted at the phagocytic cup stage by osmotic shrinking induced after the phagocytic Ca²⁺ signalling. Restoration of isotonicity was able to restore complete phagocytosis. These data thus provide evidence that the wrinkled neutrophil surface topography provides the membrane reservoir to increase the available cell surface area for phagocytosis and spreading by neutrophils.     

The behavior of human neutrophils during flow through capillary pores

The passage times of individual human neutrophils through single capillary-sized pores in polycarbonate membranes were measured with the resistive pulse technique, and results were compared to those obtained from the micropipette aspiration of entire cells. Pore transit measurement serves as a useful means to screen populations of cells, and allows for protocols that measure time dependent changes to the population. Neutrophils exhibited a highly linear pressure/flow rate relationship at aspiration pressures from 200 Pa to 1,500 Pa in both the pore and pipette systems. Cellular viscosity, as determined by the method of Hochmuth and Needham, was 89.0 Pa.s for the pore systems and 134.9 Pa.s for the pipette systems. These results are in general agreement with recent values of neutrophil viscosity published in the literature. Extrapolation of the observed linear flow response revealed an apparent minimum pressure for whole cell aspiration significantly above the threshold pressure predicted by Evans' liquid drop model. However, whole cell aspiration was achieved in both the pore and pipette systems at pressures below this extrapolated minimum, although the calculated cellular viscosity was greatly increased. The implications of these two regimes of cell deformation is unclear. This behavior could be explained by shear thinning of the material in the cell body. However the origin of this phenomenon may be in the cortical region of the cell, which exhibits an elastic tension that may be deformation rate dependent.     

Organic anion transport in macrophage membrane vesicles

Cells of the J774 mouse macrophage-like cell line possess organic anion transporter that transport fluorescent dyes such as Lucifer Yellow out of the cytoplasmic matrix of the cells; the dye is both sequestered in endosomes and secreted into the extracellular medium. Lucifer Yellow that is sequestered within endosomes is subsequently delivered to the lysosomal compartment. In the present studies we demonstrated that probenecid inhibited removal of Lucifer Yellow from the soluble cytoplasm and sequestration into membrane bound organelles by quantitating Lucifer Yellow fluorescence in both soluble and membrane-associated fractions of J774 cells. In addition, we examined the uptake of Lucifer Yellow into isolated subcellular organelles derived from J774 cells. Lucifer Yellow transport in the organellar fraction of J774 cell homogenates was temperature- and pH-dependent and did not require ATP. Subcellular organelles from J774 cells were fractionated into endosome- and lysosome-enriched fractions by Percoll density gradient centrifugation. Lucifer Yellow was preferentially taken up by vesicles of the endosome-enriched fraction, and this transport was inhibited by probenecid. These studies provide direct evidence that probenecid inhibits Lucifer Yellow transport out of the cytoplasmic matrix and into cytoplasmic vacuoles in J774 cells and that organic anion transport in isolated organelles derived from J774 cells occurs preferentially in endosome, rather than in lysosome-enriched fractions; they suggest that Lucifer Yellow is carried across membranes via a secondary active transport process that requires proton symptom or hydroxyl anion antiport.