Experimental and modelling studies of the chemoattractant-induced actin polymerization response of polymorphonuclear leukocytes
Polymorphonuclear leukocytes (PMNs) have been used in the study of cell locomotion at both the cellular and intracellular levels. However, a gap remains between observations of cell locomotion at the cellular level and our understanding of events occurring at the intracellular level. One of the important and necessary intracellular events involved in cell motility is the polymerization of the protein actin. Actin in the cell exchanges between pools of monomer (G-actin) and pools of polymeric filaments (F-actin). The state of actin within the cell is mediated by the polymerization kinetics of the protein itself and by other proteins that associate with actin (actin-binding proteins). When PMNs are stimulated with chemoattractant, they rapidly polymerize actin. We have examined the regulation of the actin state within PMNs in response to chemotactic peptides in a combination of experimental and modeling studies. We have described several techniques for studying the actin polymerization response of PMNs: (1) we characterize the binding of TRITC-phalloidin (a fluorescently labeled molecule which binds selectively to F-actin and stabilizes F-actin) to cellular F-actin, (2) we describe conditions under which TRITC-phalloidin can be used to quantify cellular F-actin in lysates without fixation and without altering the F-actin levels, and (3) we describe experiments to characterize actin filament lengths that pellet under various centrifugation conditions. These results are used in a series of experiments to measure the actin filament length distribution in stimulated and unstimulated PMNs. We analyzed F-actin depolymerization time courses in lysates (prepared in high salt buffers) from unstimulated and stimulated cells and determined that chemoattractant stimulation increased the actin filament number in PMNs from 1.7 $\pm$ 0.4 $\times$ 10$\sp5$ to 4.0 $\pm$ 0.5 $\times$ 10$\sp5$ filaments/cell. In both cases, the filament length distributions were similar with most ($\sim$80%) of the filaments $\sim$0.18 $\mu$m in length. In addition, we examined mechanisms that may be responsible for stabilization of F-actin in cell lysates. Finally, we have developed a kinetic model for the chemoattractant-induced actin polymerization response of PMNs. We have used this model to examine experimental data that are available in the literature for different aspects of the response. By studying the regulation of actin within a cell, we have begun to elucidate some of the intracellular events involved in the cell locomotion response.
Chemical engineering|Cellular biology
Cano, Manuel Luis, "Experimental and modelling studies of the chemoattractant-induced actin polymerization response of polymorphonuclear leukocytes" (1991). Dissertations available from ProQuest. AAI9211916.