Robert M. Hochmuth

Micropipette aspiration of living cells.

The mechanical behavior of living cells is studied with micropipette suction in which the surface of a cell is aspirated into a small glass tube while tracking the leading edge of its surface. Such edges can be tracked in a light microscope to an accuracy of +/-25 nm and suction pressures as small as 0.1-0.2 pN/microm2 can be imposed on the cell. Both soft cells, such as neutrophils and red cells, and more rigid cells, such as chondrocytes and endothelial cells, are studied with this technique. Interpretation of the measurements with basic continuum models leads to values for a cells elastic and viscous properties. In particular, neutrophils are found to behave as a liquid drop with a cortical (surface) tension of about 30 pN/microm and a viscosity on the order of 100 Pa s. On the other hand, chondrocytes and endothelial cells behave as solids with an elastic modulus of the order of 500 pN/microm2 (0.5 kPa).

Measurement of the Elastic Modulus for Red Cell Membrane Using a Fluid Mechanical Technique

Red cells which adhere to a surface in a parallel plate flow channel are stretched when acted on by a fluid shear stress. Three types of stretching are studied: whole cell stretching, the stretching of a red cell evagination, and tether (long, thin membrane process) stretching. In addition, the stretching of a large scale model cell attached to a surface is studied in a Couette flow channel. The results indicate that the uniaxial stretching of red cell membrane can be described by a linear stress-strain relationship. Simple theories developed from free body diagrams permit the calculation of a value for the modulus of elasticity of cell membrane in each of the three experiments. In all cases the value for the modulus is on the order of 10(4) dyn/cm(2) for an assumed membrane thickness of 0.01 mum. It was also observed that red cell tethers steadily increase in length when the fluid shear stress is greater than approximately 1.5 dyn/cm(2) and tether lengths in excess of 200 mum have been achieved. Tethers appear to possess both fluid and elastic properties.

Electro-mechanical permeabilization of lipid vesicles. Role of membrane tension and compressibility.

A simple micropipet technique was used to determine the critical electric field strength for membrane breakdown as a function of the applied membrane tension for three different reconstituted membranes: stearoyloleoylphosphatidylcholine (SOPC), red blood cell (RBC) lipid extract, and SOPC cholesterol (CHOL), 1:1. For these membranes the elastic area expansivity modulus increases from approximately 200 to 600 dyn/cm, and the tension at lysis increases from 5.7 to 13.2 dyn/cm, i.e., the membranes become more cohesive with increasing cholesterol content. The critical membrane voltage, Vc, required for breakdown was also found to increase with increasing cholesterol from 1.1 to 1.8 V at zero membrane tension. We have modeled the behavior in terms of the bilayer expansivity. Membrane area can be increased by either tensile or electrocompressive stresses. Both can store elastic energy in the membrane and eventually cause breakdown at a critical area dilation or critical energy. The model predicts a relation between tension and voltage at breakdown and this relation is verified experimentally for the three reconstituted membrane systems studied here.

Red cell extensional recovery and the determination of membrane viscosity.

A theory of membrane viscoelasticity developed by Evans and Hochmuth in 1976 is used to analyze the time-dependent recovery of an elongated cell. Before release, the elongated cell is the static equilibrium where external forces are balanced by membrane elastic force resultants. Upon release, the cell recovers its initial shape with a time-dependent exponential behavior characteristic of the viscoelastic solid model. It is shown that the model describes the time-dependent recovery process very well for a time constant in the range of 0.1-0.13 s. The time constant is the ratio membrane surface viscosity eta:membrane surface elasticity mu. Measurements for the shear modulus mu of 0.006 dyne/cm give a value for the surface viscosity of red cell membrane as a viscoelastic solid material of eta = mu tc = (6-8) X 10(-4) poise . cm.

Myosin I Contributes to the Generation of Resting Cortical Tension

The amoeboid myosin Is are required for cellular cortical functions such as pseudopod formation and macropinocytosis, as demonstrated by the finding that Dictyostelium cells overexpressing or lacking one or more of these actin-based motors are defective in these processes. Defects in these processes are concomitant with changes in the actin-filled cortex of various Dictyostelium myosin I mutants. Given that the amoeboid myosin Is possess both actin- and membrane-binding domains, the mutant phenotypes could be due to alterations in the generation and/or regulation of cell cortical tension. This has been directly tested by analyzing mutant Dictyostelium that either lacks or overexpresses various myosin Is, using micropipette aspiration techniques. Dictyostelium cells lacking only one myosin I have normal levels of cortical tension. However, myosin I double mutants have significantly reduced (50%) cortical tension, and those that mildly overexpress an amoeboid myosin I exhibit increased cortical tension. Treatment of either type of mutant with the lectin concanavalin A (ConA) that cross-links surface receptors results in significant increases in cortical tension, suggesting that the contractile activity of these myosin Is is not controlled by this stimulus. These results demonstrate that myosin Is work cooperatively to contribute substantially to the generation of resting cortical tension that is required for efficient cell migration and macropinocytosis.

Micropipette suction for measuring piconewton forces of adhesion and tether formation from neutrophil membranes

A new method for measuring piconewton-scale forces that employs micropipette suction is presented here. Spherical cells or beads are used directly as force transducers, and forces as small as 10-20 pN can be imposed. When the transducer is stationary in the pipette, the force is simply the product of the suction pressure and the cross-sectional area of the pipette minus a small correction for the narrow gap that exists between the transducer and the pipette wall. When the transducer is moving along the pipette, the force on it is corrected by a factor that is proportional to the ratio of its velocity relative to its drag-free velocity. With this technique, the minimum force required to form a membrane tether from neutrophils is determined (45 pN), and the length of the microvilli on the surface of neutrophils is inferred. The strength of this technique is in its simplicity and its ability to measure forces between cells without requiring a separate theory or a calibration against an external standard and without requiring the use of a solid surface.

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.

Mechanochemical Properties of Membranes

Publisher Summary This chapter deals with the mechanochemical properties of membranes. It intends to describe briefly the intrinsic material forces, deformation, and rate of deformation of the membrane as a continuum. From thermodynamic principles, this chapter develops the relationship among the intrinsic forces, the deformation, and rate of deformation. This provides the basis for evaluating past experimental studies on membrane mechanics, leads to a first-order appraisal of the constitutive relations among the intensive material parameters. Continuum mechanical properties are related to the molecular structure and provide direct assessment of the material arrangement. In conjunction with thermodynamic considerations, mechanochemical equations of state and material thermodynamic relations can be investigated. Furthermore, experiments must be carefully designed to measure the independent material properties of the membrane. Finally, this chapter concludes that there is a significant requirement for more experimental data.

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.

A solid-liquid composite model of the red cell membrane.

SummaryDirect mechanical experiments and analyses support the view that the red cell membrane is a composite with a solid structural matrix, which can behave as either a viscoelastic or viscoplastic material.