Single membrane tether extraction from adult and neonatal dermal microvascular endothelial cells

Membrane tethers were found to be extracted from leukocytes and macrovascular endothelial cells (e.g., human umbilical vein endothelial cells or HUVECs) when a point pulling force was exerted. These tethers stabilize leukocyte rolling on the endothelium during the inflammatory response. However, little is known about tether extraction from other vascular cells like microvascular endothelial cells (MECs). In this study, we extracted tethers from both adult and neonatal dermal MECs with the micropipette aspiration technique. We found a linear relationship between the pulling force and tether growth velocity for both cell lines. This constitutive relationship is mainly determined by the membrane mechanical property and the underlying actin-based cytoskeleton for both attached and suspended endothelial cells. It is independent of cell surface receptor type, attachment state, cytokine stimulation, or cell lineage. For both types of MECs, the threshold forces are 50 pN and the effective viscosities are around 0.5 pN·s/µm. These results, which are close to what was obtained from HUVECs, indicate that homogeneity is preserved in terms of tether extraction among different types of endothelial cells, and simultaneous tethers are likely extracted when leukocytes roll on either microvascular or macrovascular surfaces. leukocyte rolling; cell mechanics; micropipette; cytoskeleton     

A simple, rapid, aseptic method for filling micropipettes by centrifugation

A centrifugation method for sterilizing, storing, and filling micropipettes is described. Each micropipette is held in a centrifuge tube by a rubber stopper which clamps the butt end of the micropipette. The pipettes are sterilized and aseptically stored. A pipette is filled by injecting solution into the butt end of the micropipette. The micropipette is returned to a suspended position in the centrifuge tube and the liquid is rapidly forced into the micropipette tip by centrifugation. The technique is simpler and more rapid than presently used centrifugation methods.     

Determination of thermodynamics and kinetics of RNA reactions by force

Single-molecule methods have made it possible to apply force to an individual RNA molecule. Two beads are attached to the RNA; one is on a micropipette, the other is in a laser trap. The force on the RNA and the distance between the beads are measured. Force can change the equilibrium and the rate of any reaction in which the product has a different extension from the reactant. This review describes use of laser tweezers to measure thermodynamics and kinetics of unfolding/refolding RNA. For a reversible reaction the work directly provides the free energy; for irreversible reactions the free energy is obtained from the distribution of work values. The rate constants for the folding and unfolding reactions can be measured by several methods. The effect of pulling rate on the distribution of force-unfolding values leads to rate constants for unfolding. Hopping of the RNA between folded and unfolded states at constant force provides both unfolding and folding rates. Force-jumps and force-drops, similar to the temperature jump method, provide direct measurement of reaction rates over a wide range of forces. The advantages of applying force and using single-molecule methods are discussed. These methods, for example, allow reactions to be studied in non-denaturing solvents at physiological temperatures; they also simplify analysis of kinetic mechanisms because only one intermediate at a time is present. Unfolding of RNA in biological cells by helicases, or ribosomes, has similarities to unfolding by force.     

The application of a homogeneous half-space model in the analysis of endothelial cell micropipette measurements

Experimental studies have shown that endothelial cells which have been exposed to shear stress maintain a flattened and elongated shape after detachment. Their mechanical properties, which are studied using the micropipette experiments, are influenced by the level as well as the duration of the shear stress. In the present paper, we analyze these mechanical properties with the aid of two mathematical models suggested by the micropipette technique and by the geometry peculiar to these cells in their detached post-exposure state. The two models differ in their treatment of the contact zone between the cell and the micropipette. The main results are expressions for an effective Young's modulus for the cells, which are used in conjunction with the micropipette data to determine an effective Young's modulus for bovine endothelial cells, and to discuss the dependence of this modulus upon exposure to shear stress.     

Determination of the Poisson's ratio of the cell: recovery properties of chondrocytes after release from complete micropipette aspiration

Chondrocytes in articular cartilage are regularly subjected to compression and recovery due to dynamic loading of the joint. Previous studies have investigated the elastic and viscoelastic properties of chondrocytes using micropipette aspiration techniques, but in order to calculate cell properties, these studies have generally assumed that cells are incompressible with a Poisson's ratio of 0.5. The goal of this study was to measure the Poisson's ratio and recovery properties of the chondrocyte by combining theoretical modeling with experimental measures of complete cellular aspiration and release from a micropipette. Chondrocytes isolated from non-osteoarthritic and osteoarthritic cartilage were fully aspirated into a micropipette and allowed to reach mechanical equilibrium. Cells were then extruded from the micropipette and cell volume and morphology were measured throughout the experiment. This experimental procedure was simulated with finite element analysis, modeling the chondrocyte as either a compressible two-mode viscoelastic solid, or as a biphasic viscoelastic material. By fitting the experimental data to the theoretically predicted cell response, the Poisson's ratio and the viscoelastic recovery properties of the cell were determined. The Poisson's ratio of chondrocytes was found to be 0.38 for non-osteoarthritic cartilage and 0.36 for osteoarthritic chondrocytes (no significant difference). Osteoarthritic chondrocytes showed an increased recovery time following full aspiration. In contrast to previous assumptions, these findings suggest that chondrocytes are compressible, consistent with previous studies showing cell volume changes with compression of the extracellular matrix.     

Mechanical anchoring strength of L-selectin, beta2 integrins, and CD45 to neutrophil cytoskeleton and membrane.

The strength of anchoring of transmembrane receptors to cytoskeleton and membrane is important in cell adhesion and cell migration. With micropipette suction, we applied pulling forces to human neutrophils adhering to latex beads that were coated with antibodies to CD62L (L-selectin), CD18 (beta2 integrins), or CD45. In each case, the adhesion frequency between the neutrophil and bead was low, and our Monte Carlo simulation indicates that only a single bond was probably involved in every adhesion event. When the adhesion between the neutrophil and bead was ruptured, it was very likely that receptors were extracted from neutrophil surfaces. We found that it took 1-2 s to extract an L-selectin at a force range of 25-45 pN, 1-4 s to extract a beta2 integrin at a force range of 60-130 pN, and 1-11 s to extract a CD45 at a force range of 35-85 pN. Our results strongly support the conclusion that, during neutrophil rolling, L-selectin is unbound from its ligand when the adhesion between neutrophils and endothelium is ruptured.     

An Apparatus for Measuring Volumes of Small Objects

An apparatus for measuring volumes of small objects such as tissue blocks is described. The apparatus measures volumes by fluid displacement and consists of a micropipette adapted to fit the mouth of an Erleiuneyer flask, a Luer adaptor fused to the side of the flask, and a glass syringe. When assembled with fluid enclosed, the fluid rises to a low level in the micropipette. Withdrawal of fluid into the syringe lowers the fluid level below the mouth of the flask. The micropipette is raised, the object to be measured is placed in the flask, and the micropipette is joined to the flask again. Fluid returned to the flask from the syringe rises to a higher level in the micropipette. The difference between the two fluid levels equals the volume of the object measured.

This apparatus gives reproducible measurements and can be calibrated for absolute volume determination. It is inexpensive to construct and easy to use.     

Lipid-assisted microinjection: introducing material into the cytosol and membranes of small cells.

The microinjection of synthetic molecules, proteins, and nucleic acids into the cytosol of living cells is a powerful technique in cell biology. However, the insertion of a glass micropipette into the cell is a potentially damaging event, which presents significant problems, especially for small mammalian cells (spherical diameter = 2-15 micron), especially if they are only loosely adherent. The current technique is therefore limited to cells that are both sufficiently large or robust and firmly attached to a substrate. We describe here a modification of the standard technique that overcomes some of the problems associated with conventional microinjection but that does not involve the insertion of a micropipette deep into the cell cytoplasm. Instead, this method depends on lipid fusion at the micropipette tip to form a continuous but temporary conductance pathway between the interiors of the micropipette and cell. This technique thus also provides a novel method of transferring lipids and lipid-associated molecules to the plasma membrane of cells.     

A three-dimensional random network model of the cytoskeleton and its role in mechanotransduction and nucleus deformation

We have developed a three-dimensional random network model of the intracellular actin cytoskeleton and have used it to study the role of the cytoskeleton in mechanotransduction and nucleus deformation. We use the model to predict the deformation of the nucleus when mechanical stresses applied on the plasma membrane are propagated through the random cytoskeletal network to the nucleus membrane. We found that our results agree with previous experiments utilizing micropipette pulling. Therefore, we propose that stress propagation through the random cytoskeletal network can be a mechanism to effect nucleus deformation, without invoking any biochemical signaling activity. Using our model, we also predict how nucleus strain and its relative displacement within the cytosol vary with varying concentrations of actin filaments and actin-binding proteins. We find that nucleus strain varies in a sigmoidal manner with actin filament concentration, while there exists an optimal concentration of actin-binding proteins that maximize nucleus displacement. We provide a theoretical analysis for these nonlinearities in terms of the connectivity of the random cytoskeletal network. Finally, we discuss laser ablation experiments that can be performed to validate these results in order to advance our understanding of the role of the cytoskeleton in mechanotransduction.     

Optical evaluation of red blood cell geometry using micropipette aspiration.

Although red blood cell (RBC) geometry has been extensively studied by micropipette aspiration, the small size of RBC and pipettes vs. the optical resolution of light microscopy suggests the need to consider potential errors. The present study addressed such difficulties and investigated four specific problems: (1) use of exact equations to calculate RBC membrane area and volume; (2) calibration of the pipette internal diameter (PID); (3) correction for a noncylindrical pipette barrel; (4) diffraction distortion of the RBC image. The observed PID represents a cylinder lens enlargement that can be theoretically derived from the glass/buffer refractive index ratio (1.49/1.33 = 1.12). This enlargement was experimentally confirmed by: (1) studying pipettes bent to allow measurement through the barrel (horizontal) and at the orifice (vertical), with a resulting diameter ratio of 1.12 +/- 0.01; (2) and by replacing the surrounding buffer with immersion oil and hence abolishing the lens phenomenon (ratio = 1.12 +/- 0.02). In addition, use of aspirated oil droplets demonstrated a 3.2 +/- 0.2% error when the PID is focused at a sharp, maximum diameter. The average pipette cone angle was 1.49 +/- 0.09 degrees and varied considerably with pipette pulling procedures; calculated tongue geometry inside the pipette was affected by the noncylindrical pipette barrel. The RBC diffraction error, demonstrated by touching two aspirated cells held by opposing pipettes, was 0.091 +/- 0.002 microns. The PID, cone angle, and diffraction artifacts significantly (p < 0.001) affected calculated RBC geometry (average errors up to 5.4% for area and 9.6% for volume). Two new methods to calculate, rather than directly measure, the PID from images of a single RBC, during either osmotic or pressure manipulation, were evaluated; the osmotic method closely predicted the PID, whereas the pressure method markedly underestimated the PID. Our results thus confirm the need to consider the above-mentioned phenomena when determining RBC geometric parameters via micropipette aspiration.     

An application of the micropipette technique to the measurement of the mechanical properties of cultured bovine aortic endothelial cells

The mechanical properties of endothelial cells were measured using the micropipette technique. The cells employed were collected from bovine aortic endothelium and cultured in our laboratory. Endothelial cells from confluent monolayers under no-flow conditions were detached from their substrate by trypsin or by a mechanical method and suspended in modified Dulbecco medium (MDM). In the micropipette technique, a part of the cell is aspirated into the tip of the micropipette under a microscope, and the deformation measured from a photograph. In this study, the data obtained were analyzed using a model where the cytoskeletal elements, which are considered to be the primary stress bearing components, are assumed to reside in a submembranous, cortical layer. Detached cells were found to have almost homogeneous mechanical properties based on measurements from different regions of the surface of a single cell. However, a hysteresis loop was observed in the relation between pressure and cell deformation during the loading and unloading processes. The calculated elastic shear moduli obtained for the trypsin-detached cells were as much as 10-20 times larger than those of a red blood cell. Mechanically-detached cells had moduli approximately twice that of the trypsin detached cells. Passage time, i.e., cell culture age, had no influence on the mechanical properties of the trypsin-detached cells, but did have an effect on the mechanically-detached cells, with both the younger and older cells being somewhat stiffer.     

Microapplicator and micropipette for inoculation of the embryonated chicken egg.

Aflatoxin G2 was used to test the speed and accuracy of the microapplicator and the micropipette. The 50% lethal dose of both assay systems was approximately the same, and 4.0 mug of G2 had an 85% lethal effect in both systems. The 50% lethal dose of the microapplicator and the micropipette was lower than that of the syringe but, of these, only the micropipette can combine the accuracy of the microapplicator and the speed of the syringe.     

Microapplicator and micropipette for inoculation of the embryonated chicken egg.

Aflatoxin G2 was used to test the speed and accuracy of the microapplicator and the micropipette. The 50% lethal dose of both assay systems was approximately the same, and 4.0 mug of G2 had an 85% lethal effect in both systems. The 50% lethal dose of the microapplicator and the micropipette was lower than that of the syringe but, of these, only the micropipette can combine the accuracy of the microapplicator and the speed of the syringe.     

Estimation of viscous dissipation inside an erythrocyte during aspirational entry into a micropipette.

Viscous dissipation inside the erythrocyte during its aspirational entry into a micropipette is analyzed. The motion of the intracellular fluid is approximated by a flow into the micropipette orifice from a half space (the portion of the erythrocyte outside the micropipette). The stream function and intracellular pressure (p) in the half space are obtained as a function of radial and axial positions near the orifice. Solution of the boundary value problem for a uniform stream entering a circular hole gives p = 2 eta HQ/pi R3p, where eta H is the intracellular viscosity, Q is the total discharge, and Rp is the pipette radius. The results indicate that the moving erythrocyte membrane helps to drive the intracellular fluid into the orifice. For normal erythrocytes, p is only approximately 0.5% of the total aspiration pressure (delta P). The contribution of p to delta P, however, may become significant when there is a large increase in eta H due to a markedly elevated intracellular hemoglobin concentration or an alteration of the physical state of hemoglobin.     

A power-law rheology-based finite element model for single cell deformation

Physical forces can elicit complex time- and space-dependent deformations in living cells. These deformations at the subcellular level are difficult to measure but can be estimated using computational approaches such as finite element (FE) simulation. Existing FE models predominantly treat cells as spring-dashpot viscoelastic materials, while broad experimental data are now lending support to the power-law rheology (PLR) model. Here, we developed a large deformation FE model that incorporated PLR and experimentally verified this model by performing micropipette aspiration on fibroblasts under various mechanical loadings. With a single set of rheological properties, this model recapitulated the diverse micropipette aspiration data obtained using three protocols and with a range of micropipette sizes. More intriguingly, our analysis revealed that decreased pipette size leads to increased pressure gradient, potentially explaining our previous counterintuitive finding that decreased pipette size leads to increased incidence of cell blebbing and injury. Taken together, our work leads to more accurate rheological interpretation of micropipette aspiration experiments than previous models and suggests pressure gradient as a potential determinant of cell injury.     

Characterizing Cell Adhesion by Using Micropipette Aspiration

We have developed a technique to directly quantify cell-substrate adhesion force using micropipette aspiration. The micropipette is positioned perpendicular to the surface of an adherent cell and a constant-rate aspiration pressure is applied. Since the micropipette diameter and the aspiration pressure are our control parameters, we have direct knowledge of the aspiration force, whereas the cell behavior is monitored either in brightfield or interference reflection microscopy. This setup thus allows us to explore a range of geometric parameters, such as projected cell area, adhesion area, or pipette size, as well as dynamical parameters such as the loading rate. We find that cell detachment is a well-defined event occurring at a critical aspiration pressure, and that the detachment force scales with the cell adhesion area (for a given micropipette diameter and loading rate), which defines a critical stress. Taking into account the cell adhesion area, intrinsic parameters of the adhesion bonds, and the loading rate, a minimal model provides an expression for the critical stress that helps rationalize our experimental results.     

Endothelial Surface Protrusion by a Point Force

During leukocyte rolling on the endothelium, surface protrusion and membrane tether extraction occur consecutively on leukocytes. Both surface protrusion and tether extraction of leukocytes stabilize leukocyte rolling. Tethers can also be extracted from endothelial cells (ECs), but surface protrusion of ECs has never been confirmed to exist. In this study, we examined EC surface protrusion with the micropipette aspiration technique. We found that, like leukocytes, surface protrusion on an EC did exist when a point force was imposed. Both the protrusional stiffness and the crossover force of EC surface protrusion were dependent on the force loading rate and the cytoskeletal integrity, but neither of them was dependent on tumor necrosis factor α stimulation. Temperature (37°C) affected the protrusional stiffness only at small force loading rates. When a neutrophil was employed to directly impose the pulling force on the EC, simultaneous surface protrusion from both cells occurred, and it can be modeled as two springs connected in series, although the spring constants should be adjusted according to the force loading rate. Therefore, EC surface protrusion is an important aspect of leukocyte rolling, and it should not be ignored when leukocyte rolling stability is studied systematically.     

Passive deformation analysis of human leukocytes

The following analysis presents an experimental and theoretical study of the passive viscoelastic behavior of human leukocytes. Individual neutrophils in EDTA were observed both during their partial aspiration into a small micropipette and after expulsion from a large micropipette where the cell had been totally aspirated and deformed into a sausage shape. To analyze the data, a passive model of leukocyte rheology has been developed consisting of a cortical shell containing a Maxwell fluid which describes the average properties of the cell cytoplasm. The cortical shell represents a crosslinked actin layer near the surface of the cell and is assumed to be under pre-stressed tension. This model can reproduce the results of experiments using micropipette for both short-time small deformation and slow recovery data after large deformation. In addition, a finite element scheme has been established for the same model which shows close agreement with the analytical solution.     

Adhesion frequency assay for in situ kinetics analysis of cross-junctional molecular interactions at the cell-cell interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics to the binding curve returns the 2D affinity and off-rate. The assay has been validated using interactions of Fcγ receptors with IgG Fc, selectins with glycoconjugate ligands, integrins with ligands, homotypical cadherin binding, T cell receptor and coreceptor with peptide-major histocompatibility complexes. The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology, membrane anchor, molecular orientation and length, carrier stiffness, curvature, and impingement force, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules. The method has also been used to study the concurrent binding of dual receptor-ligand species, and trimolecular interactions using a modified model. The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods. To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin α(L)β(2) on neutrophils with dimeric E-selectin in the solution to activate α(L)β(2).     

An axisymmetric boundary integral model for incompressible linear viscoelasticity: application to the micropipette aspiration contact problem

The micropipette aspiration test has been used extensively in recent years as a means of quantifying cellular mechanics and molecular interactions at the microscopic scale. However, previous studies have generally modeled the cell as an infinite half-space in order to develop an analytical solution for a viscoelastic solid cell. In this study, an axisymmetric boundary integral formulation of the governing equations of incompressible linear viscoelasticity is presented and used to simulate the micropipette aspiration contact problem. The cell is idealized as a homogeneous and isotropic continuum with constitutive equation given by three-parameter (E, tau 1, tau 2) standard linear viscoelasticity. The formulation is used to develop a computational model via a "correspondence principle" in which the solution is written as the sum of a homogeneous (elastic) part and a nonhomogeneous part, which depends only on past values of the solution. Via a time-marching scheme, the solution of the viscoelastic problem is obtained by employing an elastic boundary element method with modified boundary conditions. The accuracy and convergence of the time-marching scheme are verified using an analytical solution. An incremental reformulation of the scheme is presented to facilitate the simulation of micropipette aspiration, a nonlinear contact problem. In contrast to the halfspace model (Sato et al., 1990), this computational model accounts for nonlinearities in the cell response that result from a consideration of geometric factors including the finite cell dimension (radius R), curvature of the cell boundary, evolution of the cell-micropipette contact region, and curvature of the edges of the micropipette (inner radius a, edge curvature radius epsilon). Using 60 quadratic boundary elements, a micropipette aspiration creep test with ramp time t* = 0.1 s and ramp pressure p*/E = 0.8 is simulated for the cases a/R = 0.3, 0.4, 0.5 using mean parameter values for primary chondrocytes. Comparisons to the half-space model indicate that the computational model predicts an aspiration length that is less stiff during the initial ramp response (t = 0-1 s) but more stiff at equilibrium (t = 200 s). Overall, the ramp and equilibrium predictions of aspiration length by the computational model are fairly insensitive to aspect ratio a/R but can differ from the half-space model by up to 20 percent. This computational approach may be readily extended to account for more complex geometries or inhomogeneities in cellular properties.