CELL BIOLOGY OF LOCOMOTION AND CHEMOTAXIS OF LEUKOCYTES

University Of Pennsylvania, Philadelphia PA

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Abstract

Our long term goal is to understand at a fundamental level the mechanism of chemotaxis by the polymorphonuclear leukocyte (PMN). The PMN protects the host against pathogenic bacteria. To do so, the cell detects chemical signals emanating from a site of infection, crawls toward the site, and upon reaching it, ingests and kills the bacteria. To understand the mechanism underlying this behavior is of obvious relevance to health. Earlier we studied the PMN's ability to detect a gradient of chemoattractant; we now are investigating the molecular basis for the PMN's oriented locomotion. Locomotion induced by chemoattractant is an exceedingly complex phenomenon involving many signaling pathways and many proteins. Here we focus on actin whose polymerization from the globular (G-actin) to the filamentous (F-actin) form is essential for locomotion of all cell types. F-actin levels in the PMN are regulated by chemoattractant and the induced changes in F-actin levels are sufficiently large to permit quantitative analysis. Our calculations based on the speed and magnitude of the changes indicate that there must be separate mechanisms to regulate both polymerization and depolymerization. Therefore, our goals are, to: 1) define the rates of actin turnover in vivo ; 2) localize the F-actin turnover and translocation processes in the intact cell; 3) study the factors regulating this turnover. For goal 1, we will measure the F-actin flux in resting and stimulated cells using a non-perturbing assay (exchange of 3H-adenine labeled ATP into F-actin). We will also identify factors that determine the resting F- actin level. For goal 2, we will define the rate of F-actin turnover and translocation at various positions in a locomoting cell through injection of cells with fluorescent or photoactivatable actin. We will also examine the interactions between F-actin's turnover and translocation. For goal 3, we will determine the contributions of various factors that regulate F- actin levels and dynamics. These studies will use permeabilized cells which retain their ability to increase F-actin upon stimulation with GTP- gamma-S and to decrease F-actin levels upon addition of cytochalasin. Thus, we have a functional assay which can be used to identify factors that regulate F-actin levels and turnover. Once identified, the factors will be purified from cell extracts and characterized in vitro. We anticipate that quantitative information about the rates and locations of the F-actin dynamics and identification of the molecular functions being regulated will yield a coherent mechanistic model of the PMN's chemotactic behavior.