title: F- and G-actin concentrations in lamellipodia of moving cells.
abstract: Cells protrude by polymerizing monomeric (G) into polymeric (F) actin at the tip of the lamellipodium. Actin filaments are depolymerized towards the rear of the lamellipodium in a treadmilling process, thereby supplementing a G-actin pool for a new round of polymerization. In this scenario the concentrations of F- and G-actin are principal parameters, but have hitherto not been directly determined. By comparing fluorescence intensities of bleached and unbleached regions of lamellipodia in B16-F1 mouse melanoma cells expressing EGFP-actin, before and after extraction with Triton X-100, we show that the ratio of F- to G-actin is 3.2+/-0.9. Using electron microscopy to determine the F-actin content, this ratio translates into F- and G-actin concentrations in lamellipodia of approximately 500 microM and 150 microM, respectively. The excess of G-actin, at several orders of magnitude above the critical concentrations at filament ends indicates that the polymerization rate is not limited by diffusion and is tightly controlled by polymerization/depolymerization modulators.
authors.affiliation: Institute of Molecular Biotechnology, Austrian Academy of Sciences, Vienna, Austria.
authors.forename: Stefan A
authors.initials: SA
authors.lastname: Koestler
authors.forename: Klemens
authors.initials: K
authors.lastname: Rottner
authors.forename: Frank
authors.initials: F
authors.lastname: Lai
authors.forename: Jennifer
authors.initials: J
authors.lastname: Block
authors.forename: Marlene
authors.initials: M
authors.lastname: Vinzenz
authors.forename: J Victor
authors.initials: JV
authors.lastname: Small
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry; D000199:Actins / Q000032:analysis*; D000818:Animals; D001704:Biopolymers; D045744:Cell Line, Tumor / Q000737:chemistry / Q000648:ultrastructure; D002465:Cell Movement; D049452:Green Fluorescent Proteins / Q000032:analysis; D008544:Melanocytes / Q000737:chemistry* / Q000648:ultrastructure; D008546:Melanoma, Experimental / Q000737:chemistry / Q000473:pathology; D051379:Mice; D008854:Microscopy, Electron; D009363:Neoplasm Proteins / Q000032:analysis; D038761:Photobleaching; D011554:Pseudopodia / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000032:analysis
chemical_list: D000199:Actins; D001704:Biopolymers; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins