The ability to apply controlled forces to the cell membrane may enable elucidation of the mechanisms and pathways involved in signal transduction in response to applied physical stimuli. We have developed a magnetic particle-electromagnet model that allows the application of controlled forces to the plasma membrane of substrate-attached fibroblasts. The system allows applied forces to be controlled by the magnitude of the magnetic field and by the surface area of cell membrane covered with collagen-coated ferric beads. Analysis by single-cell ratio fluorimetry of fura 2-loaded cells demonstrated large calcium transients (50-300 nM) in response to the magnetic force applications. Experiments using either the stretch-activated channel blocker gadolinium chloride or ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to eliminate external calcium ions, or addition of extracellular manganese ions, indicated that there was a calcium influx through putative stretch-activated channels. The probability of a calcium influx in single cells was increased by higher surface bead loading and the degree of cell spreading. Depolymerization of actin filaments by cytochalasin D increased the amplitude of calcium response twofold. The regulation of calcium flux by filamentous actin content and by cell spreading indicates a possible modulatory role for the cytoskeleton in channel sensitivity. Magnetic force application to beads on single cells provides a controlled model to study mechanisms and heterogeneity in physical force stimulation of cation-permeable channels.

We used two cell lines expressing fast (RPE fast ) and slow (RPE slow ) attachment kinetics to investigate mechanisms of cell-substratum adhesion. We show that the abundance of a cytoskeletal protein, vinculin, is dramatically decreased in RPE fast cells. This coincides with the diminished expression level of an endoplasmic reticulum chaperone, calreticulin. Both protein and mRNA levels for calreticulin and vinculin were decreased in RPE fast cells. After RPE fast cells were transfected with cDNA encoding calreticulin, both the expression of endoplasmic reticulum-resident calreticulin and cytoplasmic vinculin increased. The abundance of other adhesion-related proteins was not affected. RPE fast cells underexpressing calreticulin displayed a dramatic increase in the abundance of total cellular phosphotyrosine suggesting that the effects of calreticulin on cell adhesiveness may involve modulation of the activities of protein tyrosine kinases or phosphatases which may affect the stability of focal contacts. The calreticulin and vinculin underexpressing RPE fast cells lacked extensive focal contacts and adhered weakly but attached fast to the substratum. In contrast, the RPE slow cells that expressed calreticulin and vinculin abundantly developed numerous and prominent focal contacts slowly, but adhered strongly. Thus, while the calreticulin overexpressing RPE slow cells "grip" the substratum with focal contacts, calreticulin underexpressing RPE fast cells use close contacts to "stick" to it.

We have investigated in vitro the effects of the electrical field produced by constant current on freshly isolated rabbit osteoclasts and on well characterized clonal rat osteoblastlike cells. At field strengths of 0.1 and 1 V/mm, the osteoclasts migrated rapidly toward the positive electrode, whereas the osteoblastlike cells migrated in the opposite direction, toward the negative electrode. Thus, different cell types from the same tissue can respond differently to the same electrical signal. These results have important implications for hypotheses concerning the cellular mechanism of galvanotaxis, and may also clarify the cellular basis of the clinical application of electrical stimulation of bone healing.

The steady state solutions of two mathematical models are used to evaluate Munch's pressure-flow hypothesis of phloem translocation. The models assume a continuous active loading and unloading of translocate but differ in the site of loading and unloading and the route of water to the sieve tube. The dimensions of the translocation system taken are the average observed values for sugar beet and are intended to simulate translocation from a mature source leaf to an expanding sink leaf. The volume flow rate of solution along the sieve tube, water flow rate into the sieve tube, hydrostatic pressure, and concentration of sucrose in the sieve tube are obtained from a numerical computer solution of the models. The mass transfer rate, velocity of translocation, and osmotic and hydrostatic pressures are consistent with empirical findings. Owing to the resistance to water flow offered by the lateral membranes, the hydrostatic pressure generated by the osmotic pressure can be considerably less than would be predicted by the solute concentration. These models suggest that translocation at observed rates and velocities can be driven by a water potential difference between the sieve tube and surrounding tissue and are consistent with the pressure-flow hypothesis of translocation.The generation of sufficient hydrostatic pressure to overcome the resistance to solution flow offered by the sieve tube and sieve plates remains a central problem in the consideration of Munch's pressure-flow hypothesis as the mechanism of translocation in the phloem. The hydrostatic pressure available to drive solution flow has been estimated from the concentration of solutes in sieve tube sap (22,25). However, owing to resistance to water flow offered by the membranes between the sieve tube and surrounding tissue, the hydrostatic pressure in the sieve tube could be considerably less than the osmotic pressure predicted on the basis of sieve-tube-sap solute concentration.A number of mathematical models have been formulated to describe translocation in the phloem (7, 9, 11, 17; 4 for review). However, most of these models have been concerned solely with the movement of radioactive tracers (2, 9, 11) and have not dealt with the osmotic and hydrostatic pressures in sieve tubes or the movement of water into and through sieve tubes. A recent attempt to quantify these aspects of the translocation process (8) failed to deal realistically with sieve tube anatomy, including the dimensions of the sieve tube, and ignored the presence of sieve plates. In addition, translocation is a continuous process, and a model attempting to simulate translocation should include continuous loading and unloading of translocate.This paper describes two mathematical models based on irreversible thermodynamics that attempt to quantify the pressure-flow hypothesis of phloem translocation. These models can be used to predict the osmotic and hydrostatic pressure required to drive solution flow in sieve tubes and to evaluate the pressure-flow hypothesis as a plausible mecha...

We describe a new method that uses straightforward physics to apply force to substrate-attached cells. In this method, collagen-coated magnetic ferric oxide beads attach to the dorsal surface of cells via receptors of the integrin family, and a magnetic field gradient is applied to produce a force. In this paper we present a complete characterization of the method in a configuration that is easy to use, in which a permanent magnet provides a fairly uniform gradient over a relatively large area. This allows a fairly uniform average force that can be controlled in magnitude, direction, and duration to be applied to a large number of cells. We show how to determine the applied force per cell by measuring the force per unit volume of magnetic bead, the distribution of bead diameters, and the distribution of beads per cell. We also show how to calculate the force per unit volume of bead in a three-dimensional region near the permanent magnet on the basis of field measurements, and present results for three of the magnets. An upward force applied to fibroblasts by this method produces a measurable time-dependent increase in attachment of cytoskeletal actin filaments to the force application points, and an increase in actin cross-linking. This is accompanied by an actin-dependent retraction of the force-induced upward movement of the dorsal surface of the cells.