Adherence of human phagocytes results in characteristic reorganization and redistribution of distinct F-actin pools.

Abstract

The F-actin based microfilamentous cytoskeleton (MFC) provides mobility for phagocytic immune cells including polymorphonuclear leukocytes (PMNs) and macrophages (MOs). In PMNs in suspension, the MFC is organized into two distinct F-actin pools [Triton Insoluble F-actin-(TIF), which form the sub-membranous, 3D actin meshwork and Triton Soluble F-actin (TSF), which exists as short oligomers] in equilibrium with G-actin. The structure of F-actin pools in adherent cells is unknown despite the fact that phagocytes are adherent in tissues in vivo. In order to determine the structure of F-actin pools in adherent phagocytes, human PMNs were isolated and allowed to adhere to plastic for 1 hour at 37 degrees C. Adherent cells were collected, actin pools separated and quantified by SDS-PAGE and compared to nonadherent PMNs in suspension. Likewise, the nonadherent human myeloid cell line U937 was induced to MO morphology and adherence by exposure to TPA (10(-6) M x 3 days) and similarly evaluated. Adherence of PMNs to plastic resulted in 75 +/- 15% adherence (n = 3). TPA differentiation of U937 cells resulted in 81 +/- 15% adherence (n = 10). In both cells, adherence resulted in a statistically significant increase in TIF, a decrease in TSF, and little to no change in G-actin. Basal, nonadherent PMNs in suspension contain TIF 40 +/- 0%, TSF 20 +/- 4%, and G-actin 40 +/- 4%, n = 3, whereas adherent PMNs contain TIF 61 +/- 3%, TSF 5 +/- 5%, G-actin 34 +/- 1%, n = 3. Basal U937 contain TIF 41 +/- 9%, TSF 17 +/- 6%, and G-actin 42 +/- 13%, n = 7. Adherent MO-like U937 contain TIF 53 +/- 4%, TSF 9 +/- 5%, and G-actin 38 +/- 4%. The results show that phagocyte adherence leads to a characteristic reorganization of actin pool structure that is remarkably quantitatively similar to, yet mechanistically distinct from, reorganization by chemotactic factor activation in suspension. Adherence-induced TIF-actin growth results exclusively from conversion of TSF-actin to TIF-actin.