F- and G-actin concentrations in lamellipodia of moving cells.
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.
Koestler
Stefan A
SA
Institute of Molecular Biotechnology, Austrian Academy of Sciences, Vienna, Austria.
Rottner
Klemens
K
Lai
Frank
F
Block
Jennifer
J
Vinzenz
Marlene
M
Small
J Victor
JV
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
D000199:Actins; D001704:Biopolymers; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins