Actin-depolymerizing factor and cofilin-1 play overlapping roles in promoting rapid F-actin depolymerization in mammalian nonmuscle cells - PubMed (original) (raw)

Comparative Study

Actin-depolymerizing factor and cofilin-1 play overlapping roles in promoting rapid F-actin depolymerization in mammalian nonmuscle cells

Pirta Hotulainen et al. Mol Biol Cell. 2005 Feb.

Abstract

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.

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Figures

Figure 1.

Expression levels and subcellular localizations of cofilin-1 and ADF. (A) Western blot assay demonstrating the specificities of the anti-ADF and anti-cofilin-1 antibodies. Purified recombinant cofilin-1 (lane 1, 2 ng; lane 2, 8 ng; lane 3, 32 ng) and ADF (lane 4, 2 ng; lane 5, 8 ng; lane 6, 32 ng) were visualized on Western blots by using anti-cofilin-1 (top) and anti-ADF (bottom) antibodies. (B) The levels of cofilin-1 and ADF in NIH 3T3, B16F1, and Neuro 2A cells were compared with known concentrations of cofilin-1 (top) and ADF (bottom). Lane 1, 30 ng of cofilin-1 (top) and 7.5 ng of ADF (bottom); lane 2, 80 ng of cofilin-1 (top) and 20 ng of ADF (bottom); lane 3, 10 μg of NIH 3T3 extract; lane 4, 10 μg of B16F1 extract; lane 5, 10 μg of Neuro 2A extract. Cofilin-1 is expressed approximately in sixfold (NIH 3T3), 11-fold (B16F1), and sevenfold (Neuro 2A) higher molar amounts than ADF. (C) Cofilin-1, ADF, and F-actin were visualized by immunofluorescence in NIH 3T3 cells. Cofilin-1 and ADF show similar subcellular localizations and are concentrated in F-actin-rich ruffles. Bar, 10 μm.

Figure 2.

siRNA induced gene silencing of ADF or cofilin-1. (A) Western blot analysis demonstrating the ADF and/or cofilin-1 protein levels in B16F1 (lanes 1-4) and NIH 3T3 cells (lanes 5-8) transfected with control (lanes 1 and 5), ADF-specific (lanes 2 and 6), cofilin-1-specific (lanes 3 and 7), and with both ADF- and cofilin-1-specific (lanes 4 and 8) siRNA oligonucleotide duplexes. Equal amounts of cell lysates were run on polyacrylamide gels, and cofilin-1, ADF, and β-actin were visualized by Western blotting. (B-E) Cofilin-1 and ADF antibody stainings are decreased in siRNA-transfected cells. (B and C) B16F1 cells transfected with FITC-labeled cofilin-1-specific siRNA. (B) Anti-cofilin-1 antibody staining. (C) FITC-siRNA. (D and E) NIH 3T3 cells transfected with FITC-labeled ADF-specific siRNA. (D) Anti-ADF antibody staining. (E) FITC-siRNA. The borders of the transfected cells are indicated by white lines. Bars, 10 μm.

Figure 3.

Depletion of ADF or cofilin-1 resulted in an accumulation of thick actin stress fibers and an increase in cell size. B16F1 cells were treated with cofilin-1-(D-F) or ADF (G-I)-specific duplex oligonucleotides, or simultaneously with both oligonucleotides (J-L). Cofilin-1 (B, E, and K) and ADF (H) were visualized by isoform-specific antibodies and F-actin (A, C, D, F, G, I, J, and L) with rhodamine-phalloidin. (K) Both the FITC-labeled siRNA and cofilin-1 antibody staining. Representative cells from each population are indicated by white arrowheads and shown with larger magnifications (C, F, I, and L) (L is rotated 90° counterclockwise). Bars, 50 μm.

Figure 8.

G-actin/F-actin ratio is decreased in cofilin-1/ADF knockdown cells. B16F1 (A-F) and NIH 3T3 (G-L) cells were treated with cofilin-1-(A-C and G-I) or ADF (D-F and J-L)-specific duplex oligonucleotides. Cofilin-1 (A and G) and ADF (D and J) were visualized by isoform-specific antibodies, F-actin with Alexa488-phalloidin (B, E, H, and K), and G+F-actin with β-actin AC-15 antibody (C, F, I, and L). White arrows indicate the siRNA-treated cells with dramatically reduced cofilin-1/ADF levels. Bars, 10 μm. (M and N) The intensities of phalloidin and AC-15 (M) or phalloidin and DNAseI (N) stainings were analyzed from 20 ADF and cofilin-1 knockdown B16F1 and NIH 3T3 cells by TINA software, and the relative AC-15/phalloidin or DNAseI/phalloidin stainings were compared with the ones from neighboring wild-type cells to yield the ratio of (AC-15/phalloidin)knockdown/(AC-15/phalloidin)wild-type (M) or (DNAseI/phalloidin)knockdown/(DNAseI/phalloidin)wild-type (N). The knockdown cells showed a decrease in the amount of G-actin compared with nontransfected wild-type cells. SEMs are indicated in the graphs.

Figure 4.

Rescue of ADF or cofilin-1 knockdown phenotype. B16F1 and NIH 3T3 cells were treated with cofilin-1- or ADF-specific FITC-siRNA oligonucleotides followed by a transfection with myc-tagged rescue constructs refractory to siRNA. The cells transfected only with siRNA oligonucleotides (white arrows) were identified by presence of FITC-oligonucleotides (middle row) and by lack of myc-tag staining (right). These cells showed a typical knockdown phenotype with an accumulation of abnormal F-actin structures. The cells transfected with both siRNA and rescue construct (arrowheads) were identified by the simultaneous presence of FITC-oligonucleotides and anti-myc staining. These cells displayed similar actin phenotype to the nontransfected wild-type cells. It is also important to note that the ADF knockdown phenotype in NIH3T3 cells could be rescued by overexpression of cofilin-1 (bottom row). Bars, 10 μm.

Figure 5.

Cofilin-1 and ADF play overlapping roles in cytokinesis. (A and B) Wild-type and cofilin-1 or ADF knockdown NIH 3T3 cells were fixed, and DNA was visualized by DAPI staining. (A) Representative examples of a cofilin-1 knockdown cells (cell borders are indicated by white lines). Cofilin-1 was visualized with an anti-cofilin-1 antibody (left), and DNA with DAPI staining (right). (B) The number of multinucleated cells was counted from at least 700 wild-type and knockdown cells from four independent experiments. The depletion of ADF or cofilin-1 resulted in a small increase in the number of multinucleated cells, whereas the silencing of both genes resulted in a synergistic increase in the amount of multinucleated cells. (C) Time-lapse analysis of cytokinesis of wild-type (top and Supplementary Video 1) and cofilin-1 knockdown (bottom and Supplementary Video 2) NIH 3T3 cells. Frames “0 min.” represent metaphase. Black arrows indicate the positions of chromosomes. Wild-type cells undergo cell division and spreading within 30 min after the metaphase (last frame in C), whereas the process in cofilin-1 knockdown cell is significantly slower and the cell spreading is complete after ∼90 min of metaphase. Supplementary videos display the entire division processes. (D) The same cofilin-1 knockdown cell as shown in C was fixed and stained with cofilin-1 antibodies and with DAPI (first panel) to visualize the two nuclei present in the cofilin-1 knockdown cell. (E and F) Visualization of F-actin and myosin II during cytokinesis in wild-type and cofilin-1 knockdown cells. Wild-type and cofilin-1 knockdown B16F1 cells were fixed, DNA was visualized by DAPI, F-actin by phalloidin, and myosin II by an anti-myosin II antibody. Representative cells undergoing telophase (E) and late telophase (F) are shown. Bars, 20 μm.

Figure 6.

Cofilin-1 and ADF are required for cell migration. Wild-type and cofilin-1/ADF knockdown NIH 3T3 cells were plated on coverslips coated with fibronectin and blue fluorescent beads, grown for 20 h, and fixed for immunofluorescence. The wild-type cells (A) exhibited relatively long and thin phagokinetic motility tracks. In contrast, the cofilin-1 (B), ADF (C) and cofilin-1/ADF (D) knockdown cells showed clearance of beads only in their immediate vicinity and seldom displayed directional motility tracks. The borders of the cells are indicated with white lines. Bars, 20 μm. (E) The minimum motility distances were quantified from wild-type, cofilin-1, ADF, and cofilin-1/ADF knockdown cells (n ≥ 24). The silencing of cofilin-1 or ADF results in 2- to 2.5-fold decreases, and cofilin-1/ADF results in threefold decrease in the length of the phagokinetic motility tracks. SEMs are indicated in the graph.

Figure 7.

Live cell analysis of wild-type and cofilin-1 knockdown B16F1 cell migration. Wild-type B16F1 cells expressing GFP-actin (A and Supplementary Video 3) displayed fast actin dynamics in the lamellipodia and directional cell motility. Cofilin-1 knockdown cells (B and Supplementary Video 4) were unable to migrate but were still capable of slowly extending and retracting their lamellipodia. White arrows indicate the locations of the nuclei in the first frame. White arrowheads indicate largest protrusions and retractions. Bars, 10 μm. (C) Migration of 35 wild-type and 25 cofilin-1 knockdown B16F1 cells were monitored for 100 min, and the positions of the nuclei was tracked every 20 min. The average motility distances of wild-type cells are 51.0 μm and cofilin-1 knockdown cells 26.9 μm. SEMs and statistical significance of the data are indicated in the graph.

Figure 9.

FRAP analysis of actin treadmilling rates in stress fibers and lammelipodia. B16F1 cells expressing GFP-actin were treated with a cofilin-1- or ADF-specific duplex oligonucleotides and incubated for 5-12 h before analysis. The selected cell region was bleached with an intense laser beam and the fluorescence recovery was monitored by taking time-lapse images. (A and B) Actin dynamics at stress fibers. Time-lapse images were acquired every 10 s starting at 30 s after bleaching. (A) Representative examples of wild-type (top), cofilin-1 (middle), and ADF (bottom) knockdown cells before and after bleaching. Bars, 10 μm. (B) The rate of fluorescence recovery of the bleached region was analyzed with TINA software from representative wild-type (black triangles) and ADF (black squares) or cofilin-1 knockdown (open squares) cells. In each frame, the fluorescence intensity of the bleached region was compared with the fluorescence of the control region (in same picture next to bleached region) to diminish the error caused by normal photobleaching during the monitoring period. The equilibration of fluorescence between bleached and un-bleached regions in cofilin-1 knockdown cells is significantly slower than in wild-type cells. (C and D) Actin dynamics at lamellipodial regions. Time-lapse images were acquired every 3 to 4 s immediately after bleaching. (C) Representative examples of wild-type (top) and cofilin-1 knockdown cells (bottom). It is important to note that the photobleaching was carried out during the time period 3.5-37.5 s and thus the 41-s time-lapse image represents the situation at 3.5 s after the end of bleaching. Bars, 10 μm. (D) The rate of lamellipodial actin meshwork growth was quantified from wild-type and knockdown cells. Time points 0 and 3.36 show the widths of lamellipodial actin meshworks before bleaching, and the time points at 41-95 s show the widths of the lamellipodial GFP-actin meshworks after the bleaching period.

Figure 10.

Actin filament depolymerization rates in wild-type and ADF/cofilin-1 knockdown cells. Filamentous actin was visualized by phalloidin staining in wild-type (A and B), cofilin-1 knockdown (C and D), ADF knockdown (E and F), and cofilin-1/ADF knockdown (G and H) B16F1 cells after the addition 2 μM latrunculin-A. Time points 30 min after DMSO addition (control) (A, C, E, and G) and 10 min after addition of 2 μM latrunculin-A (B, D, F, and H) are shown. (I) Percentage amount of retracted cells without clear stress fibers and with abnormal F-actin aggregates were counted (n = 50 cells) at time points 5, 10, and 30 min after latrunculin-A addition. The actin filament structures were rapidly disrupted in wild-type cells (black squares). In contrast, stress fibers disappeared much more slowly in cofilin-1 (open squares) and cofilin-1/ADF (open triangles) knockdown cells. The ADF knockdown cells (black triangles) lost their actin filament structures almost as quickly as the wild-type cells. Bars, 50 μm.

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Actin-depolymerizing factor and cofilin-1 play overlapping roles in promoting rapid F-actin depolymerization in mammalian nonmuscle cells - PubMed (original) (raw)