title: Evidence for a gelsolin-rich, labile F-actin pool in human polymorphonuclear leukocytes.
abstract: Filamentous (F) actin is a major cytoskeletal element in polymorphonuclear leukocytes (PMNs) and other non-muscle cells. Exposure of PMNs to agonists causes polymerization of monomeric (G) actin to F-actin and activates motile responses. In vitro, all purified F-actin is identical. However, in vivo, the presence of multiple, diverse actin regulatory and binding proteins suggests that all F-actin within cells may not be identical. Typically, F-actin in cells is measured by either NBDphallacidin binding or as cytoskeletal associated actin in Triton-extracted cells. To determine whether the two measures of F-actin in PMNs, NBDphallacidin binding and cytoskeletal associated actin, are equivalent, a qualitative and quantitative comparison of the F-actin in basal, non-adherent endotoxin-free PMNs measured by both techniques was performed. F-actin as NBDphallacidin binding and cytoskeletal associated actin was measured in cells fixed with formaldehyde prior to cell lysis and fluorescent staining (PreFix), or in cells lysed with Triton prior to fixation (PostFix). By both techniques, F-actin in PreFix cells is higher than in PostFix cells (54.25 +/- 3.77 vs. 23.5 +/- 3.7 measured as mean fluorescent channel by NBDphallacidin binding and 70.3 +/- 3.5% vs. 47.2 +/- 3.6% of total cellular actin measured as cytoskeletal associated actin). These results show that in PMNs, Triton exposure releases a labile F-actin pool from basal cells while a stable F-actin pool is resistant to Triton exposure. Further characterizations of the distinct labile and stable F-actin pools utilizing NBDphallacidin binding, ultracentrifugation, and electron microscopy demonstrate the actin released with the labile pool is lost as filament. The subcellular localization of F-actin in the two pools is documented by fluorescent microscopy, while the distribution of the actin regulatory protein gelsolin is characterized by immunoblots with anti-gelsolin. Our studies show that at least two distinct F-actin pools coexist in endotoxin-free, basal PMNs in suspension: 1) a stable F-actin pool which is a minority of total cellular F-actin, Triton insoluble, resistant to depolymerization at 4 degrees C, gelsolin-poor, and localized to submembranous areas of the cell; and 2) a labile F-actin pool which is the majority of total cellular F-actin, Triton soluble, depolymerizes at 4 degrees C, is gelsolin-rich, and distributed diffusely throughout the cell. The results suggest that the two pools may subserve unique cytoskeletal functions within PMNs, and should be carefully considered in efforts to elucidate the mechanisms which regulate actin polymerization and depolymerization in non-muscle cells.
authors.affiliation: Department of Pediatrics, University of Alabama, Birmingham 35233.
authors.forename: R G
authors.initials: RG
authors.lastname: Watts
authors.forename: T H
authors.initials: TH
authors.lastname: Howard
mesh_terms: D000199:Actins / Q000032:analysis* / Q000378:metabolism; D000546:Amanitins / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000032:analysis*; D002451:Cell Compartmentation; D003599:Cytoskeleton / Q000648:ultrastructure; D018260:Gelsolin; D006801:Humans; D008840:Microfilament Proteins / Q000032:analysis*; D009504:Neutrophils / Q000737:chemistry*; D011108:Polymers
chemical_list: D000199:Actins; D000546:Amanitins; D002135:Calcium-Binding Proteins; D018260:Gelsolin; D008840:Microfilament Proteins; D011108:Polymers; C028877:7-nitrobenz-2-oxa-1,3-diazole-phallacidin