Cofilin is the dominant isoform compared with the actin depolymerizing factor (ADF) in several cells (Hotulainen et al., 2005
), including MTLn3 cells (Wang et al., 2004
). To suppress cofilin expression we used siRNA that specifically targets cofilin, but not ADF. Using this sequence, Western blot analysis showed that the cofilin levels were lowered by an average of 95% in MTLn3 cells, as compared with control (oligofectamine only) treated cells at 36 h after transfection (); reversion to normal levels was observed by 96 h (Mouneimne et al., 2004
). Because the antibody used for Western blotting in these experiments recognizes both cofilin and ADF, the small amount of protein remaining in cells after the siRNA treatment is consistent with the small amount of ADF relative to cofilin. We tested two other siRNA sequences as negative controls: a scrambled version of the cofilin siRNA, and a non-silencing bacterial siRNA, which does not have target sequences in MTLn3 cells. Using Western blotting and immunofluorescence, both siRNAs did not cause any change in the cofilin expression levels, whereas the cofilin siRNA significantly reduced cofilin expression ().
Figure 1. Suppression of cofilin using siRNA. (A) Western blotting of whole cell lysates of cells treated with the different siRNAs, blotted for cofilin. The western was repeated four times, and the corresponding quantification is shown in B. (C) representative (more ...)
Control, scrambled, and bacterial siRNA treated cells (, and d, respectively) showed similar morphology to that of untreated cells, whereas cofilin siRNA treated cells (referred to hereafter as CF KD) exhibited an elongated morphology (, b). Using FACS analysis, we also tested the viability of the control and CF KD cells and found that at 36 h after siRNA transfection, the majority of cells were viable (unpublished data). Therefore, all further experiments reported in the paper were performed 36 h after siRNA transfection, when maximum suppression of cofilin expression was observed (). It is important to note that suppression of cofilin did not affect the expression of other factors that are important in the regulation of the actin cytoskeleton, such as WAVE 2 and N-WASP expression (Fig. S1, C and D; available at http://www.jcb.org/cgi/content/full/jcb.200707009/DC1
Cofilin siRNA knockdown cells are characterized by an elongated shape and an increase in F-actin–containing structures
To investigate the effect of CF KD on the morphology of MTLn3 cells, we examined the ratio of length to width (L/W) of control and siRNA-treated cells. CF KD cells exhibited a two- to threefold increase in L/W ratio () compared to control cells. The scrambled and non-silencing bacterial siRNA showed similar L/W ratio as control cells. Next, we transfected the CF KD cells with a human cofilin plasmid that is not targeted by our siRNA sequence, or a control plasmid, and scored their L/W ratio. The cells transfected with the human cofilin rescue plasmid had WT-like morphology, whereas the cells transfected with the control plasmid showed high L/W ratio characteristic of CF KD cells, demonstrating that the elongated phenotype was due to cofilin suppression. Furthermore, shape analysis performed with the DIAS software demonstrated that the maximum length and perimeter of CF KD cells were almost twice that of control cells, while roundness was significantly reduced. These changes were rescued by expression of the human cofilin plasmid ().
Figure 2. Cofilin siRNA knockdown cells are elongated and show changes in F-actin structures. (A) Method of length/width (L/W) measurement. Length was scored as Feret's diameter, which is the longest distance between any two points along the boundary, black line; (more ...)
Expression of the human cofilin plasmid can rescue the phentotypic changes seen in CF KD cells
Previous studies have shown that overexpression of constitutively active LIM-kinase changes F-actin organization in MTLn3 cells, causing the formation of more prominent F-actin fibers and F-actin aggregates (Zebda et al., 2000
). To determine if the CF KD cells exhibit a similar phenotype, cells were fixed, stained using rhodamine-phalloidin, and divided into three categories as described previously (Zebda et al., 2000
) according to the structure of their actin cytoskeleton. Cells were double-blind scored, where control cells were compared with control cells, and CF KD cells were compared with other CF KD cells (representative cells from each group exhibiting the different F-actin categories are shown in ). CF KD cells had more prominent F-actin fibers and more F-actin aggregates (, white arrows) compared to control cells (). These results are consistent with the observations reported by Zebda et al. (2000)
, thus showing that lowering either cofilin expression or activity yields the same result. Recent reports have also shown that siRNA suppression of cofilin in NIH3T3 and mouse neuroblastoma cells led to an accumulation of F-actin, increase in thickness of stress fibers, and decrease in actin stress fiber turnover as measured by FRAP (Hotulainen et al., 2005
). Our analysis showed that CF KD caused a significant (P = 0.0024) increase in the total F-actin levels, presumably resulting from the loss of actin depolymerization by cofilin and hence accumulation of F-actin ().
Cofilin knockdown increases the directionality and decreases the turning frequency of MTLn3 cells
Because of the importance of cofilin in actin-based protrusions, we studied the effects of CF KD on MTLn3 cell motility. Using time-lapse microscopy, control and CF KD cells were imaged and the corresponding videos were analyzed using DIAS (Videos 1–3, available at http://www.jcb.org/cgi/content/full/jcb.200707009/DC1
). Centroid plot analysis, which shows the position of the centroids of cells traced at consecutive time intervals, showed that CF KD cells followed a more linear path compared to the random walking path, typical of MTLn3 cells and control cells (). Additionally, stacked perimeter plots revealed that CF KD cells translocated at least five to six cell body lengths in 45 min in comparison to control cells, which traversed only one to two cell body lengths. Consistent with this analysis, CF KD cells also showed significant differences in both total and net path lengths (). Consequently, directionality, which is the ratio of net to total path length, was also significantly higher in CF KD cells (). Expression of the WT(ΔH) cofilin plasmid rescued this phenotype by reducing directionality to control levels (; available at http://www.jcb.org/cgi/content/full/jcb.200707009/DC1
). These results strongly suggest that MTLn3 cells with lower cofilin levels are more directional than the control cells.
Figure 3. Cofilin knockdown cells exhibit directional migration. (A) Centroid plots of representative control and CF KD cells show the trajectory of cells over time (4 h), analyzed using DIAS. Black dots represent path of cells over time (centroid). (B) Perimeter (more ...)
To pursue this, the direction change and persistence (speed/direction change) parameters were determined using DIAS. Results demonstrated that CF KD cells have lower direction change and are simultaneously more persistent in their movement than control cells (). The frequency of turning was calculated as the angle between the trajectories that the cells were projected to follow between consecutive frames if they moved in a straight path, and the actual path they followed during the time lapse (, cartoon). Analysis showed that CF KD cells had a significantly lower turning frequency than control cells (). Consistent with their higher directionality, CF KD cells also had higher cell migration velocities than control cells because they tended to more efficiently cover distance due to less frequent turns (). Overall, similar results were obtained whether cell movements were imaged for 45 min, 4 h, or 12 h, indicating that subtle changes in cell shape do not contribute to the analysis of directionality, persistence, and turning frequency (unpublished data).
In support of the CF KD phenotype in MTLn3 cells, analysis of LIMK overexpressing MTLn3 (F) cells showed that these cells have an elongated morphology and enhanced directionality similar to CF KD cells (Fig. S3, A–E; available at http://www.jcb.org/cgi/content/full/jcb.200707009/DC1
). F cells also showed a directional motility behavior similar to the CF KD cells (Fig. S3, B, D, and d) when compared with control cells (Fig. S3, C and c) and cells expressing the kinase-dead domain of LIMK (KS) (Fig. S3, E and e). This illustrates that inactivating cofilin has the same general phenotype as reducing its expression.
Cofilin knockdown phenotype in mesenchymal-like non metastatic tumor cells is different from that in metastatic tumor cells
Using the same siRNA, we were able to knockdown cofilin levels ~80% in MTC, nonmetastatic mammary adenocarcinoma cells (). MTC cells have a mesenchymal (fibroblast-like) type of motility exhibiting a well-developed unipolar leading edge with a large amount of F-actin and a characteristic rectilinear movement (Shestakova et al., 1999
). Suppression of cofilin in MTC cells led to the formation of multipolar lamellipodia () and caused a decrease in the directionality of migration consistent with the appearance of multiple lamellipodia (). These results suggest that cofilin is involved in the maintenance of directionality of migration in mesenchymal-like non-metastatic tumor cells, like that previously described for fibroblasts (Dawe et al., 2003
), as compared with decreasing directionality by increasing turning frequency in metastatic amoeboid type tumor cells as described above for MTLn3 cells ().
Figure 4. Cofilin knockdown changes the motility behavior of MTC cells. (A) Western blot of whole cell lysate samples taken from MTC cells treated either with oligofectamine (control), or cofilin siRNA (CF KD). (B) quantification of the levels of cofilin in MTC (more ...)
CF KD increases the persistence and stability, and decreases both the frequency and rate, of protrusions in MTLn3 cells
To understand the contributions of changes in protrusion to the phenotype observed in metastatic tumor cells following CF KD, we measured the protrusion dynamics in control and CF KD cells. Using shape and kymograph analysis we found that the protrusions in CF KD cells were concentrated in just a few areas of the cell membrane, whereas in control cells protrusions were observed with equal frequency around the entire perimeter of the cell (). Using DIAS, we followed protrusions (green) and retractions (red) in time-lapse videos (, arrows) and determined the frequency and persistence of these events. CF KD cells had more persistent but less frequent protrusions than control cells (). Additionally, the number of protrusions lasting for 10 min was similar in both groups; whereas CF KD cells tend to have more protrusions lasting for longer time intervals ().
Figure 5. Importance of cofilin to the dynamics of protrusions in MTLn3 cells. (A) shape analysis of representative control, and CF KD cells, taken from a time lapse recorded over 4 h (y-axis), shown as unwrapped stacked shapes of cells. Unwrapping was done by (more ...)
Using kymography, we found that CF KD cells have protrusions of approximately the same size as control cells () but that are more persistent () and with lower velocities (). Expression of the WT(ΔH) cofilin plasmid in CF KD cells was able to rescue these changes seen in CF KD cells (Fig. S2, E–G; available at http://www.jcb.org/cgi/content/full/jcb.200707009/DC1
). Combined, these results suggest that cofilin is important in inducing the formation of globally distributed protrusions with higher velocity in MTLn3 cells, and in helping to turn over the protrusions once formed.
Cofilin knockdown in MTLn3 cells inhibits chemotaxis by desensitizing the cell perimeter to directional stimulation
Amoeboid chemotaxis is a hallmark of metastatic mammary tumor cells like MTLn3 (Wyckoff et al., 2007
). Thus, we studied the EGF-induced motility and chemotaxis of CF KD MTLn3 cells because EGF is a well-documented chemotactic stimulus in these cells (Mouneimne et al., 2004
). Control and CF KD cells were starved for 3–4 h, and then were bath stimulated with 5 nM EGF. F-actin accumulation at the leading edge (), and the rate of protrusion were suppressed in CF KD cells (; Fig. S4, and Videos 4–6; available at http://www.jcb.org/cgi/content/full/jcb.200707009/DC1
). The initial rate of lamellipod protrusion was rescued in CF KD cells that were cotransfected with the human cofilin plasmid (; Fig. S4 and Videos 4–6). These results are consistent with the lower turning rate of CF KD cells because lamellipod extension is a first step in the assignment of cell direction (Mouneimne et al., 2004
) and fewer lamellipods per time might decrease turning frequency.
Figure 6. Cofilin knockdown suppresses EGF-induced F-actin assembly and protrusion at the leading edge and the ability of the cells to chemotaxis. (A) representative control and CF KD GFP-actin expressing MTLn3 cells, over a range of 6 min; 0 min (no stimulation), (more ...)
We used tropomyosin staining to determine if loss of cofilin perturbs or abolishes the lamellipodial actin network without affecting the lamellar network. Previous reports using quantitative fluorescent speckle microscopy in migrating epithelial cells found that there are two distinct F-actin regions; the lamellipodium which consists of a dynamic F-actin network and has high concentrations of Arp2/3 complex and cofilin, and the lamella which has myosin II and tropomyosin (Gupton et al., 2005
). Tropomyosin has been shown to be a useful marker for distinguishing the lamellipod and lamella compartments (DesMarais et al., 2002
). In this context, we analyzed the localization of tropomyosin, and found that CF KD did not abolish the lamellipod compartment (Fig. S4, B and C), consistent with previous work indicating that cofilin alone is not responsible for lamellipod formation (Mouneimne et al., 2004
The ability of local activation of cofilin to initiate protrusion as described previously (Ghosh et al., 2004
), and the requirement of cofilin for cell turning as described here, suggest that cofilin activity is required for protrusion which in turn is required for turning, i.e., setting cell direction. In other words, to chemotax efficiently a cell must be able to respond to chemotactic stimulation at any region on its surface and to turn in response to changes in direction of the chemotactic stimulus. To investigate this, cells were divided into front and back, as defined by the position of the pipette. In control cells this was random because there was no fixed front and back, whereas in CF KD cells the front of the cell was taken as the leading edge of the elongated cells (). Control cells exhibited similar sensitivity on all sides of the cell toward a pipette delivering a gradient of EGF (). However, CF KD cells were only able to protrude toward the pipette when it was at the front of the cell, but retracted away from the pipette when stimulated at the back (). These results indicate that cofilin is required for the ability of a cell to protrude uniformly around its periphery when stimulated with EGF.
The elongated phenotype of CF KD cells was intriguing in its resemblance to the elongated cell shape resulting from the enrichment of myosin II in the uropods of streaming Dictyostelium
cells and migrating neutrophils (Zhang et al., 2002
). Polarized myosin II localization to the uropod has been hypothesized to specify the rear of the cell and antagonize protrusion activity there. To investigate if myosin II is localized to the rear of CF KD cells thereby causing the elongation and unipolar protrusion phenotype, we stained CF KD MTLn3 cells for myosin IIA and IIB isoforms. As shown in Fig. S5 (available at http://www.jcb.org/cgi/content/full/jcb.200707009/DC1
), myosin II is uniformly distributed in control and slightly concentrated in the front of CF KD cells. No accumulation of myosin II was detected in the rear of these elongated cells, and there was no difference between total myosin II levels between control and CF KD cells. Thus, the phenotype of CF KD cells could not be explained by myosin II accumulation at the rear of the cells.
Cofilin knockdown causes relocalization of Arp2/3 to one side of MTLn3 cells
Because cofilin generates newly polymerized actin filaments that are preferred for dendritic nucleation by the Arp2/3 complex in vitro (Ichetovkin et al., 2002
), we investigated whether CF KD produced any change in the localization of the Arp2/3 complex in the cell. Interestingly, our results showed that CF KD led to the asymmetric localization of the Arp2/3 complex to the front of the elongated cell (). These results also showed that in control cells the level of Arp2/3 complex is the same in randomly chosen front and back regions.
Figure 7. Cofilin knockdown causes relocalization of Arp2/3 to the front of the elongated MTLn3 cells. (A) Arp2/3 staining of control, and CF KD cells. Insets show the front (f) and the back (b) at higher magnification; asterisks represent the regions that are (more ...)
To determine if the asymmetric distribution of Arp2/3 complex in CF KD cells contributed to their elongated shape and directional migration, we suppressed the expression of Arp2/3 complex by siRNA knock down of the p34 subunit. This is sufficient to decrease the expression of other subunits of the complex (Fig. S1, F–H) and the activity of the complex (Kempiak et al., 2003
). Although p34 knockdown (referred to hereafter as Arp KD) did not affect the shape and directionality of control cells, Arp KD reverted the elongated shape and directionality of CF KD cells to control levels ().
Figure 8. Arp2/3 complex is required for the shape and direction of migration of metastatic MTLn3 cells. (A) Representative images of control (i), CF KD (ii), Arp KD (iii), and DB KD (iv). Bar, 10 μm. (B) Quantification of the L/W of the groups in A. Number (more ...)
Additionally, we studied the rate of protrusion, stimulated by 5 nM EGF, of Arp KD and DB KD cells (CF KD in which p34 is also knocked down) and found that both types of treatments generated cells that show significant decreases in their protrusion in response to EGF (). However, Arp KD and DB KD cells were still able to protrude. The observation of protrusion activity in Arp KD and DB KD cells is consistent with previous work showing that Arp3-siRNA treated cells are still able to generate protrusions and migrate (Di Nardo et al., 2005
) and the ability of p34 knockdown cells to protrude lamellipodia in MTLn3 cells in particular (unpublished data).
DIAS analysis of movies of DB KD cells (in serum) also showed reversion to the normal random-walking motility behavior and turning frequency of MTLn3 cells similar to that seen in control cells () suggesting that Arp2/3 complex contributes to the directionality and decreased turning frequency seen in CF KD cells. Arp KD and DB KD cells also showed lowered cell migration velocities indicating that optimum cell migration requires the Arp2/3 complex (). Interestingly, Arp KD and DB KD cells did not show an increase in prominent F-actin structures like that seen in CF KD cells but had a significant increase in F-actin aggregates compared with control cells, suggesting that F-actin dynamics are altered in Arp KD cells ().
Using kymography, we found that the absence of Arp2/3 complex produced a slight decrease in protrusion velocity compared with CF KD cells (), whereas protrusions from both Arp KD and DBKD cells were more persistent () and larger () than in control or CF KD cells. This latter result is consistent with increased protrusion size seen in MTLn3 cells resulting from activation of mDia proteins after inhibition of Arp2/3 complex activity (unpublished data).
Effects of activated Rac on the cofilin knock down phenotype
Regulation of the actin cytoskeleton by the Rho family of GTPases is an important factor in the regulation of cell protrusion dynamics (Nobes and Hall, 1995
). By pull-down assays in CF KD cells, we found that there was no change in the activity of the Rho GTPases (unpublished data). Previous work on MTLn3 cells has shown that constitutively active Rac results in global lamellipod protrusion (El-Sibai et al., 2007
), and that Rac is not required for the initiation of protrusions, but is for lamellipodial stability (Yip et al., 2007
). Thus, we investigated if constitutively active Rac is able to affect the shape and motility phenotypes observed in CF KD cells. We found that the transfection of CF KD cells with a constitutively active Rac plasmid (GFP-RacQ61L) rescued their elongated phenotype (). Cells expressing the plasmid had lower length to width ratio (, red arrowheads) than CF KD cells that were not expressing the plasmid (, white arrowheads) resulting from the return to apolar protrusion activity. CF KD cells expressing the Rac mutant also showed reversion of the increase in speed seen in CF KD cells consistent with the renewed ability to protrude in all directions. GFP-RacQ61L expression increased the size and persistence of protrusions beyond that resulting from CF KD () consistent with previous findings that Rac stabilizes protrusions in MTLn3 cells (Yip et al., 2007
). In addition, the affects of activated Rac on the behavior of CF KD cells is consistent with a role for Arp2/3 complex in the motility phenotypes of CF KD cells.
Figure 9. Activated Rac rescues the elongated phenotype of the cofilin siRNA knockdown cells. (A) Constitutively active GFP-RacQ61L was able to rescue the elongated phenotype of the CF KD cells. (A) L/W ratio of control and CF KD cells transfected with GFP-RacQ61L. (more ...)