Neutrophil crawling is an important physiological and pathological activity. During crawling the cell's cytoskeleton undergoes rapid changes in its molecular organization, often resulting in pseudopod formation through rapid addition of actin monomers in a local region along the cell membrane. We have developed an assay whereby neutrophils are stimulated by micropipet manipulation to form pseudopod-like structures. This assay is a new method for studying the kinetics of cell cytoskeleton rearrangement. A latex bead with coupled antibody for the adhesion receptor CD18 is used to make a point contact with the neutrophil surface. A tether is then formed by pulling the cell away from the bead, which triggers the formation of actin network from the cell body. This network advances along the tether at a constant velocity. This velocity is limited by the slowest rate of the processes involved in network formation, i.e., either the transport of mass (diffusion or convection) or the slowest of the chemical reactions. We have already shown that the rate of network formation is not limited by diffusion or polymerization of actin. A possible candidate for the rate limiting factor is convection mass transport, which depends on cytoplasm viscosity. Both cytoplasm viscosity and rates of the chemical reactions involved in network formation depend on temperature. This temperature dependence is used to determine whether or not convection mass transport is the rate limiting factor in network formation. To do this, the Arrhenius plot of the velocity of network advancement is compared to the same plot of the cytoplasm viscosity. In the former case, the slope of the Arrhenius plot depends on the concentration of calcium in both the bathing solution and the cell cytoplasm, while in the latter case, the slope of the Arrhenius plot is independent of calcium concentration. This shows that convection mass transport is not the rate limiting factor in network formation. Most probably, the rate limiting factor is one of the chemical reactions involved in the dissociation of actin from its capping proteins or is related to the formation of nucleation sites. In future experiments, we will examine the effects on network formation in tethers and pseudopods due to stimulation by a variety of chemoattractants.
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