Actin-depolymerizing factor and cofilin-1 play overlapping roles in promoting rapid F-actin depolymerization in mammalian nonmuscle cells.
Actin-depolymerizing factor (ADF)/cofilins are small actin-binding proteins found in all eukaryotes. In vitro, ADF/cofilins promote actin dynamics by depolymerizing and severing actin filaments. However, whether ADF/cofilins contribute to actin dynamics in cells by disassembling "old" actin filaments or by promoting actin filament assembly through their severing activity is a matter of controversy. Analysis of mammalian ADF/cofilins is further complicated by the presence of multiple isoforms, which may contribute to actin dynamics by different mechanisms. We show that two isoforms, ADF and cofilin-1, are expressed in mouse NIH 3T3, B16F1, and Neuro 2A cells. Depleting cofilin-1 and/or ADF by siRNA leads to an accumulation of F-actin and to an increase in cell size. Cofilin-1 and ADF seem to play overlapping roles in cells, because the knockdown phenotype of either protein could be rescued by overexpression of the other one. Cofilin-1 and ADF knockdown cells also had defects in cell motility and cytokinesis, and these defects were most pronounced when both ADF and cofilin-1 were depleted. Fluorescence recovery after photobleaching analysis and studies with an actin monomer-sequestering drug, latrunculin-A, demonstrated that these phenotypes arose from diminished actin filament depolymerization rates. These data suggest that mammalian ADF and cofilin-1 promote cytoskeletal dynamics by depolymerizing actin filaments and that this activity is critical for several processes such as cytokinesis and cell motility.
Hotulainen
Pirta
P
Program in Cellular Biotechnology, Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland. pirta.hotulainen@helsinki.fi
Paunola
Eija
E
Vartiainen
Maria K
MK
Lappalainen
Pekka
P
D000199:Actins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D048429:Cell Size; D051338:Cofilin 1; D048749:Cytokinesis / Q000235:genetics; D051345:Destrin; D005347:Fibroblasts / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate; D036681:Fluorescence Recovery After Photobleaching; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D020868:Gene Silencing; D007211:Indoles; D007700:Kinetics; D008546:Melanoma, Experimental / Q000378:metabolism*; D051379:Mice; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D018715:Microscopy, Video; D041681:NIH 3T3 Cells; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D022502:Stress Fibers / Q000378:metabolism; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines
D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; C495811:Cfl1 protein, mouse; D051338:Cofilin 1; D051345:Destrin; C495825:Dstn protein, mouse; D005353:Fibronectins; D005456:Fluorescent Dyes; D007211:Indoles; D008840:Microfilament Proteins; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D013844:Thiazoles; D053778:Thiazolidines; C007293:DAPI; D016650:Fluorescein-5-isothiocyanate; C037067:latrunculin A