Quantitation of Total F-actin in Swiss 3T3 Cells during Lamellipodium and Stress Fiber Formation
Serum starvation of confluent quiescent Swiss 3T3 cells causes the loss of stress fibers, actin-rich lamellipodia, and membrane ruffles (24
). Using a modified version of the rhodamine phalloidin binding assay of Howard and Wang (15
), we found that serum starvation for 18 h also reduced the F-actin content of cells by ~50% (data not shown). This depolymerization could provide a pool of actin monomer in starved cells so that a burst of actin polymerization might occur when cells are induced to form stress fibers and/or lamellipodia by activation of Rac or Rho. We measured F-actin levels in cells before and after stimulation with sphingosine-1-p to activate Rho, PDGF to activate Rac, sphingosine-1-p plus PDGF to activate both Rac and Rho, or serum to simulate a return to normal growing conditions. All treatments increased the F-actin levels somewhat (Fig. ). Sphingosine-1-p addition (Fig. , black bars
) caused a transient rise to 22% above resting levels of F-actin, which had decreased to 12% above background by 60 min. PDGF addition (Fig. , dark shaded bars
) caused a somewhat larger and more sustained response to a maximum of 28% above baseline, which decreased back to 20% by 60 min. Within 5 min of addition, sphingosine-1-p plus PDGF (Fig. , light shaded bars
) or serum (Fig. , white bars
) completely restored F-actin levels to near prestarvation values and sustained this increased level for 60 min.
All treatments resulted in most of the cells responding appropriately (stress fibers or ruffles or some combination). Treatment of cells with sphingosine-1-p or serum resulted in stress fiber formation in 100% of the cells over the time course, whereas ruffling was more variable, and more difficult to quantify, as sizes of lamellipodia varied widely. At least 500 cells were counted for each point. Representative cells from each time point are shown in Fig. . Fig. a shows starved cells, which do not contain stress fibers, and <5% of the cells show any ruffles. Within 5 min of PDGF addition, 93% of cells show extensive ruffling (not shown), which is sustained for at least 20 min (91% at 10 min, 89% at 20 min; Fig. b). By 60 min, only 16% of cells are ruffling (Fig. c). This shows a rough correlation with the time course of the changes in F-actin levels in response to PDGF shown in Fig. , although the decrease in ruffling is clearly more dramatic than the F-actin change. In the first 5 min of sphingosine-1-p addition, 100% of cells show a loose bundling of actin filaments (Fig. d), which thicken and become straighter during a 60-min time course (Fig. , e and f); fewer than 3% of cells showed any ruffles. Addition of 10% serum to starved cells results in a sustained increase in the proportion of cells ruffling. At 5 min after addition of serum, 44% of the cells are ruffling (Fig. g). By 10 min, 82% are ruffling, and at 20 min, 87% of the cells are still ruffling (Fig. h). By 60 min 77% of the cells continue to show ruffling (Fig. i). In addition to ruffling, 100% of the cells exposed to 10% serum form stress fibers with approximately the same time course as we observed after sphingosine-1-p addition. Addition of both sphingosine-1-p and PDGF to cells largely mimics the addition of serum, with all cells forming stress fibers and a transient increase in the proportion of cells ruffling (5 min, 51%, 10 min, 59%, 20 min, 32%, and 60 min, 18%; Fig. , j, k, and l, respectively).
Figure 2 Examples of the cellular response to stimuli used in the quantitation of F-actin experiments. All photos show rhodamine phalloidin labeling of Swiss 3T3 cells taken directly from the F-actin quantitation of Fig. . (a) Typical serum-starved (more ...)
Actin Dynamics Are Affected by the Activation State of Small GTPases
The incorporation of Cy3-actin into F-actin–containing structures in growing cells is likely to be at least partially due to the activation state of the small GTPases Rac and Rho. Comicroinjection of Cy3-actin and C3 transferase (to inactivate Rho) had no effect on incorporation of Cy3- actin into lamellipodia even though stress fibers were largely disassembled in these cells after 20 min (Fig. , a and a′). Similarly, coinjection of dominant negative Rac protein with Cy3-actin, which blocked lamellipodia extension and peripheral Cy3-actin incorporation, did not affect the slow rate of incorporation of Cy3-actin into stress fibers (Fig. , b and b′). In serum-starved cells, Cy3-actin remained largely diffuse in the cell body for at least 20 min (Fig. , c and c′). This suggests that Rac and Rho exert separate controls on actin dynamics in the cell, and that in serum-starved conditions, when Rac and Rho have low activity, minimal localized actin polymerization or turnover occurs.
Figure 3 Cy3-actin polymerization is influenced by Rac and Rho activation states. Swiss 3T3 cells growing in 10% serum (a, a′, b, and b′) or serum starved overnight (c and c′) were microinjected with Cy3-actin and inhibitors of Rho (C3-transferase; (more ...)
Actin Polymerization in Rac and PDGF-induced Lamellipodia
To determine whether actin polymerization is induced by Rac in Swiss 3T3 cells, recombinant constitutively active Rac protein and Cy3-actin monomer were coinjected into serum-starved Swiss 3T3 cells. Within 5 min of injection, polymerization of Cy3-actin could be observed in the periphery of injected cells (Fig. , a [Cy3-actin] and a′ [fluorescein phalloidin counterstain]). The F-actin–containing lamellipodium expanded in injected cells and, by 20–25 min, large ruffling lamellipodia were present that contained Cy3-actin (Fig. , b [Cy3-actin] and b′ [fluorescein phalloidin counterstain]).
Figure 4 Incorporation of Cy3-actin into ruffling lamellipodia induced by activation of Rac. Serum-starved confluent quiescent Swiss 3T3 cells were microinjected with Cy3-actin and constitutively active Rac protein (a, a′, b, and b′) or treated (more ...)
Cy3-actin also rapidly incorporated into ruffling lamellipodia when starved cells were first injected with Cy3-actin and then treated with PDGF. Within 5 min of PDGF treatment, lamellipodia contained concentrated Cy3-actin (Fig. , c [Cy3-actin] and c′ [fluorescein phalloidin counterstain]). At 20–25 min, lamellipodia showed similar localization of Cy3-actin as with recombinant Rac injection (Fig. , d [Cy3-actin] and d′ [fluorescein phalloidin counterstain]).
To determine quantitatively whether PDGF addition had essentially the same effect as microinjection of recombinant active Rac, we compared the amount of Cy3-actin incorporated into lamellipodia produced by each treatment. To do this, we photographed at least 20 cells coinjected with a mixture of Cy3-actin, fluorescein dextran, and, for the Rac experiments, constitutively active Rac protein. We compared the intensity of Cy3-actin fluorescence with the level of dextran fluorescence in the lamellipodium to obtain an estimate of how much Cy3-actin had polymerized in the lamellipodium (above levels that would be expected because of cell thickness in this region). An example of the data we obtained is shown in Fig. . In each graph (a–d) the fluorescence intensity profile of either Cy3-actin or fluorescein dextran is shown as a function of distance across the cell. Cells treated with PDGF (not shown) or injected with Rac (Fig. , c and d) showed relatively larger peripheral fluorescence peaks of Cy3- actin than did starved cells (Fig. , a and b). The ratios of fluorescence intensities are shown in Fig. e. Both endogenous and recombinant Rac show identical time courses of activation, with Cy3-actin becoming concentrated in the lamellipodium by 5 min of injection or PDGF treatment. The amount of Cy3-actin accumulation after PDGF treatment was smaller than that after microinjected active Rac (twofold for Rac, Fig. e, white bars; 1.5-fold for PDGF, Fig. e, gray bars) and stayed largely constant for both treatments over a time course of 5–20 min. Starved cells showed little or no concentration of Cy3-actin in the periphery (Fig. e, black bars). We also summed the intensities of all Cy3-actin peaks obtained after either Rac injection or PDGF treatment and found no significant size differences over time with the different treatments (data not shown). Thus, both activation of endogenous Rac by PDGF treatment and microinjection of recombinant activated Rac produce a rapid polymerization and accumulation of actin in peripheral lamellipodia. In addition, since the total intensity of the Cy3-actin fluorescence in the lamellipodium does not increase over the time course, turnover of F-actin must also be similar in the two treatments.
Localized Actin Polymerization Does Not Accompany Rho-induced Stress Fiber Formation
To understand how actin is assembled into stress fibers, we used confocal imaging of de novo stress fiber formation at different times after activation of Rho with sphingosine-1-p. Starved cells contain diffuse and punctate phalloidin staining, as well as actin filament bundles loosely organized on the bottom surface of the cell and around the nucleus. They also typically contain a thick peripheral bundle of actin filaments and sometimes rings of actin filaments that are distributed throughout the cell, primarily on the basal surface (Fig. a, arrow and inset). These structures appear to be bundled actin filaments, which stain with myosin-II antibodies and incorporate Cy3-actin with a half-time of ~20 min (not shown). Electron microscope studies of whole mount preparations of starved cells have shown that the rings are not separate structures, but sites of tight coiling of curvilinear actin bundles (Rottner, K., and J.V. Small, personal communication). By 5 min after sphingosine-1-p addition, most cells show increased aggregation of actin filament bundles (Fig. b). These cells also show diffuse phalloidin staining suggestive of nonbundled actin filaments that are below the resolution of the light microscope. By 20 min, parallel arrays of stress fibers (Fig. c) (with focal adhesions; not shown) appear in most cells, and these thicken and lengthen by 60 min of treatment (Fig. d).
Figure 6 Time course of formation of stress fibers upon Rho activation with sphingosine-1-p. Serum-starved confluent quiescent Swiss 3T3 cells were examined by confocal microscopy to determine the organization of filamentous actin stained with rhodamine phalloidin (more ...)
We next examined incorporation of monomeric Cy3-actin into stress fibers after activation of Rho. After addition of sphingosine-1-p for 5 min to confluent quiescent cells, actin bundles are present (Fig. a′) even though these are relatively thinner than later stress fibers and poorly organized. They are not detectably labeled with Cy3-actin (Fig. a). By 20 min after sphingosine-1-p treatment, some incorporation of Cy3-actin into stress fibers occurs (Fig. , b and b′). At 40 min after treatment, most of the Cy3-actin incorporates into the stress fibers in most cells (Fig. , c and c′). Coinjection of dominant active Rho protein into cells also causes stress fibers to form, which are initially unlabeled with Cy3-actin (10 min after injection; Fig. , d and d′) but gradually incorporate the labeled actin as turnover occurs (20 min after injection; Fig. , e and e′), with nearly complete incorporation by 40 min (not shown).
Figure 7 Time course of incorporation of Cy3-actin into stress fibers induced by Rho activation. Serum-starved confluent quiescent Swiss 3T3 cells were treated in two ways to cause Rho activation: either injection of Cy3-actin and then addition of sphingosine-1-p (more ...)
Role of Extracellular Matrix in Rho-induced Assembly of Stress Fibers and Focal Adhesions
When serum-starved cells are trypsinized and replated on poly-l
-lysine, they do not form focal adhesions or stress fibers (13
). Fig. , a
, shows, however, that addition of sphingosine-1-p to cells plated on poly-l
-lysine induces a dramatic reorganization of filamentous actin; after stimulation, the periphery of cells is nearly devoid of phalloidin label and the actin is assembed into disorganized bundles in the cell body. Myosin also moves from diffuse cytoplasmic and cortical labeling to colocalize with the actin bundles in the body of the cell (Fig. , c
). Coinjected Cy3-actin only gradually incorporates into these actin bundles, with a half-time of ~20 min (data not shown), similar to incorporation rates for stress fibers. Interestingly, vinculin can be found concentrated along the length of these actin-myosin cables (Fig. , e
), but there are no focal adhesion plaques, as there is no extracellular matrix in these conditions. There is also no visible recruitment of talin to these structures (Fig. , g
) and very little (if any) concentration of paxillin (not shown). In contrast, Rho activation in cells plated on fibronectin causes the assembly of parallel actin stress fibers with both talin and paxillin concentrated in focal adhesions (visualized with the same antibodies; data not shown). Actin-myosin assemblies formed upon Rho activation in cells plated on poly-l
-lysine do appear to exert tension, as cells show dramatic rounding, e.g., when sphingosine-1-p is added to cells, as observed under time-lapse video microscopy (data not shown).
Figure 8 Response of cells plated on poly-l-lysine to Rho activation. Serum-starved quiescent Swiss 3T3 cells were trypsinized and plated onto poly-l-lysine (10 μg/ml)–coated coverslips for 20 min before addition of sphingosine-1-p for 20 min (more ...)
To rule out that the accumulation of vinculin along Rho-induced actin bundles in cells on poly-l-lysine was due to small or transient integrin clustering, we used EDTA treatment concomitantly with sphingosine-1-p addition to dissociate integrin complexes. We found that the number of cells containing vinculin clusters formed by Rho activation on poly-l-lysine, and the morphology of these clusters was completely resistant to 1 mM or 10 mM EDTA treatment, whereas 100% of the cells on fibronectin rounded up and lost focal adhesions under these conditions (data not shown).
Role of Extracellular Matrix in Rac-induced Lamellipodium Extension
To determine whether integrin-mediated adhesion is required for the extension of lamellipodia and ruffles, Rac and Cy3-actin were coinjected into cells plated on poly-l
-lysine (Fig. , a
), or PDGF was used to activate endogenous Rac in cells plated on poly-l
-lysine (Fig. , c
) or Con A (not shown), two substrates that do not lead to integrin clustering. Within 5 min (not shown) and for up to 20 min (Fig. a
), Cy3-actin accumulated in the expanding lamellipodium of cells plated on poly-l
-lysine and colocalized with F-actin as shown by counterstaining with fluorescein phalloidin (Fig. b
). These lamellipodia appear enriched in vinculin (Fig. c
; same cell counterstained with rhodamine phalloidin, Fig. d
), but they have no detectable organized focal complexes like those seen on fibronectin (Fig. e
; same cell counterstained with rhodamine phalloidin, Fig. f
). Rac- and PDGF-induced lamellipodia in cells plated on poly-l
-lysine also appear wider in proportion to the cell body size and more uniform around the periphery of the cell than lamellipodia induced in cells plated on fibronectin (Fig. f
). Using video microscopy, we found that the cells retained the ability to ruffle on poly- l
-lysine in a manner largely indistinguishable from cells on fibronectin-coated surfaces (not shown). Cells plated on Con A–coated coverslips also extend lamellipodia in response to microinjection of activated Rac protein, and ruffles can be observed by time-lapse video that are indistinguishable from those formed on fibronectin substrates (not shown). Thus Rac-induced focal complexes are not important for lamellipodium extension, ruffling, or actin polymerization in the lamellipodium.
Figure 9 Response of cells plated on poly-l-lysine to Rac activation. Serum-starved quiescent Swiss 3T3 cells were trypsinized and plated onto poly-l-lysine (10 μg/ ml)–coated coverslips for 20 min (a–d) or fibronectin-coated coverslips (more ...)