Role of gelsolin in the formation and organization of triton-soluble F-actin during myeloid differentiation of HL-60 cells.
Structurally and functionally distinct F-actin pools coexist with globular (G)-actin in a variety of eukaryotic cells, including polymorphonuclear leukocytes (PMNs). In PMNs, a Triton-soluble F-actin pool (TSF) exists as short cytoplasmic filaments capped with gelsolin, while Triton-insoluble F-actin (TIF) is a three-dimensional meshwork of F-actin associated with actin-binding protein 280 (ABP-280), alpha-actinin, and tropomyosin. The unique association of gelsolin with the TSF suggests a role for gelsolin in creation or regulation of TSF. To evaluate gelsolin's role in TSF formation, the quantities of actin and gelsolin were determined by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblots in uninduced HL-60 cells (U-HL-60) and in HL-60 cells induced to myeloid differentiation with 1.25% dimethyl sulfoxide for 4 to 5 days (I-HL-60). U-HL-60 cells contain 17.76 +/- 6.01 pmol actin per 10(6) cells (TIF, 5.3 +/- 1.5; TSF, 2.17 +/- 0.37; G, 10.3 +/- 5.7; n = 5) and 0.073 pmol gelsolin per 10(6) cells (TIF, 0; TSF, 0.002 +/- 0.005; G, 0.07 +/- 0.01; n = 3), representing molar actin to gelsolin (A:G) ratios of 1,085:1 for TSF and 147:1 for G. After myeloid differentiation, the actin content increases 1.80-fold (31.94 +/- 6.14 pmol/10(6) cells) equally in each actin pool (TIF, 9.36 +/- 2.35; TSF, 3.29 +/- 0.62; G, 19.29 +/- 4.83). Gelsolin increases 2.4-fold overall (0.178 +/- 0.02 pmol/10(6) cells) but 19-fold in TSF (0.038 +/- 0.009) and only 1.9-fold in G pool (0.139 +/- 0.006), resulting in A:G ratios of 87:1 in TSF and 139:1 in G. The findings of an increase in TSF gelsolin with decreased A:G ratios (1,085:1 v 87:1) with myeloid differentiation suggest shortening of TSF filaments, while the A:G ratios of unbound gelsolin are unchanged (147:1 v 139:1). Measurement of EGTA-resistant gelsolin/actin complexes in HL-60 cells shows that 95% to 100% of complexes exist in the TSF-actin pool only. These findings are consistent with a role for gelsolin in formation and organization of Triton-soluble F-actin. Furthermore, the apparent shortening of TSF-actin filaments with myeloid cellular differentiation and maturation may represent one mechanism of conversion of the nonmotile myeloblast to the motile PMN.
Watts
R G
RG
Department of Pediatrics, University of Alabama at Birmingham 35233, USA.
D000199:Actins / Q000378:metabolism*; D001704:Biopolymers / Q000378:metabolism; D002451:Cell Compartmentation; D002454:Cell Differentiation / Q000187:drug effects; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D003285:Contractile Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000502:physiology / Q000648:ultrastructure; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D064448:Filamins; D018260:Gelsolin / Q000502:physiology*; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000378:metabolism / Q000473:pathology*; D008840:Microfilament Proteins / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000502:physiology*; D017830:Octoxynol; D016897:Respiratory Burst; D012995:Solubility; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000199:Actins; D001704:Biopolymers; D003285:Contractile Proteins; C578661:FLNC protein, human; D064448:Filamins; D018260:Gelsolin; D008840:Microfilament Proteins; D009363:Neoplasm Proteins; D009240:N-Formylmethionine Leucyl-Phenylalanine; D017830:Octoxynol; D013755:Tetradecanoylphorbol Acetate; D004121:Dimethyl Sulfoxide