title: Mechanisms responsible for F-actin stabilization after lysis of polymorphonuclear leukocytes.
abstract: While actin polymerization and depolymerization are both essential for cell movement, few studies have focused on actin depolymerization. In vivo, depolymerization can occur exceedingly rapidly and in a spatially defined manner: the F-actin in the lamellipodia depolymerizes in 30 s after chemoattractant removal (Cassimeris, L., H. McNeill, and S. H. Zigmond. 1990. J. Cell Biol. 110:1067-1075). To begin to understand the regulation of F-actin depolymerization, we have examined F-actin depolymerization in lysates of polymorphonuclear leukocytes (PMNs). Surprisingly, much of the cell F-actin, measured with a TRITC-phalloidin-binding assay, was stable after lysis in a physiological salt buffer (0.15 M KCl): approximately 50% of the F-actin did not depolymerize even after 18 h. This stable F-actin included lamellar F-actin which could still be visualized one hour after lysis by staining with TRITC-phalloidin and by EM. We investigated the basis for this stability. In lysates with cell concentrations greater than 10(7) cells/ml, sufficient globular actin (G-actin) was present to result in a net increase in F-actin. However, the F-actin stability was not solely because of the presence of free G-actin since addition of DNase I to the lysate did not increase the F-actin loss. Nor did it appear to be because of barbed end capping factors since cell lysates provided sites for barbed end polymerization of exogenous added actin. The stable F-actin existed in a macromolecular complex that pelleted at low gravitational forces. Increasing the salt concentration of the lysis buffer decreased the amount of F-actin that pelleted at low gravitational forces and increased the amount of F-actin that depolymerized. Various actin-binding and cross-linking proteins such as tropomyosin, alpha-actinin, and actin-binding protein pelleted with the stable F-actin. In addition, we found that alpha-actinin, a filament cross-linking protein, inhibited the rate of pyrenyl F-actin depolymerization. These results suggested that actin cross-linking proteins may contribute to the stability of cellular actin after lysis. The activity of crosslinkers may be regulated in vivo to allow rapid turnover of lamellipodia F-actin.
authors.affiliation: Department of Biology, University of Pennsylvania, Philadelphia 19104-6018.
authors.forename: M L
authors.initials: ML
authors.lastname: Cano
authors.forename: L
authors.initials: L
authors.lastname: Cassimeris
authors.forename: M
authors.initials: M
authors.lastname: Fechheimer
authors.forename: S H
authors.initials: SH
authors.lastname: Zigmond
mesh_terms: D000185:Actinin / Q000032:analysis / Q000494:pharmacology; D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis; D003599:Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D007700:Kinetics; D008840:Microfilament Proteins / Q000032:analysis; D009504:Neutrophils / Q000378:metabolism* / Q000648:ultrastructure; D010590:Phalloidine; D011108:Polymers; D011189:Potassium Chloride / Q000494:pharmacology; D011554:Pseudopodia / Q000378:metabolism; D011817:Rabbits; D014335:Tropomyosin / Q000032:analysis
chemical_list: D000199:Actins; D002352:Carrier Proteins; C034037:F-actin-binding proteins; D008840:Microfilament Proteins; D011108:Polymers; D014335:Tropomyosin; D000185:Actinin; D010590:Phalloidine; D011189:Potassium Chloride