Changes in Cell Morphology That Accompany Cell Spreading on Fibronectin
When grown in culture, many cells form specialized adhesive structures that link them to the underlying matrix. During the assembly of these structures, there are dramatic changes in cell morphology. To define the changes in cell morphology that accompany cell adhesion and spreading, serum-starved Rat1 fibroblasts were plated onto fibronectin for various times and stained for focal adhesions and F-actin. Fig. shows 15-min and 60-min time points. Rat1 cells adhered rapidly to fibronectin, with >90% of the cells adhering within 10 min. By 15 min, most of the cells had begun to flatten, with large membranous protrusions (“ruffles”) appearing at the cell perimeter (Fig. a
). In those cells that had initiated cell spreading, punctate concentrations of vinculin were present at the perimeter in structures (Fig. b
) that resembled “point contacts” (Tawil et al., 1993
) or “focal complexes” (Nobes and Hall, 1995
). F-actin was also present at the cell perimeter in membranous protrusions that appeared to be membrane ruffles (Fig. a
). At this time there were very few cytoplasmic actin filaments that could be visualized. However, at 60 min there were many bundles of actin filaments that spanned the central core of the cell (Fig. c
), and these bundles appeared to be anchored at the cell periphery in vinculin-containing focal adhesions (Fig. d
Figure 1 Assembly of focal adhesions and stress fibers during adhesion and spreading of Rat1 fibroblasts on fibronectin. Serum-starved Rat1 fibroblasts (a–d) or cells expressing dominant-inhibitory Cdc42 (e–h), Rac1 (I–L), or RhoA ( (more ...)
These structures that formed when cells adhered and spread on an insoluble ECM protein such as fibronectin closely resembled the structures that form when adherent Swiss 3T3 cells are stimulated with PDGF or serum (Ridley et al., 1992
; Ridley and Hall, 1992
). To test whether Rat1 cells were also susceptible to activation by these soluble factors, serum-starved adherent Rat1 cells were treated with buffer, PDGF, or FBS for 10 min, and then stained for F-actin. As shown in Fig. a
, some F-actin–containing structures remained in serum-starved Rat1 cells, but serum treatment of these cells resulted in a dramatic increase in actin bundles (Fig. c
) and PDGF treatment induced the localization of F-actin into membrane ruffles (Fig. b
). Therefore, both soluble and insoluble factors are capable of stimulating the formation of cytoskeletal structures such as membrane ruffles, focal adhesions, and actin bundles in Rat1 cells.
Figure 2 PDGF and serum induce the assembly of membrane ruffles and stress fibers in serum-starved adherent Rat1 cells. Parental (a–c), dominant-inhibitory Rac- (d), or Rho-expressing (e) Rat1 cells were plated on fibronectin-coated coverslips in serum-free (more ...)
Cell Spreading Is Cdc42, Rac, and Rho Dependent
Whereas members of the Rho family of small GTPases are known to regulate the assembly of cytoskeletal structures stimulated by soluble factors, relatively little is known about the role of the Rho family in integrin-mediated changes in cell morphology. To examine this, we used Rat1 cells that inducibly express dominant-inhibitory mutants of the Rho family members Cdc42, Rac1, and RhoA (Fig. ). The Rat1 cell lines selected for this study expressed the highest levels of N17Cdc42, N17Rac, and N19Rho; they have been previously characterized (Qiu et al., 1995a
; Qiu et al., 1997
). The levels of expression of the dominant-inhibitory mutants were sufficient to (1
) inhibit PDGF-induced membrane ruffling in N17Rac-expressing cells (Fig. d
), and (2
) inhibit stress fiber formation induced by serum in N19Rho-expressing cells (Fig. e
Figure 3 Expression of dominant- inhibitory mutants of Rho-family members Cdc42, Rac1, and RhoA in Rat1 cells. Rat1 fibroblasts (lane 1) or cells expressing a Myc epitope–tagged N17Cdc42 (lane 2), N17Rac1 (lane 3), or N19RhoA (lane 4) were grown for (more ...)
We examined if the Rho-family members Cdc42, Rac, and/or Rho control a cell's ability to adhere to and/or spread on a matrix. Rat1 cells expressing dominant-inhibitory mutants of Cdc42, Rac, or Rho were plated onto fibronectin for 20 min, fixed, stained, and then the number of spread and nonspread cells counted. No significant differences between Rat1 cells and dominant-inhibitory Cdc42-, Rac-, or Rho-expressing cells were detected in the numbers of cells that adhered to fibronectin (data not shown). However, a significant reduction in cell spreading was observed in cells expressing dominant-inhibitory Cdc42, Rac, or Rho (Fig. ).
Figure 4 Rho, Rac, and Cdc42 are required for Rat1 cells to spread on fibronectin. Serum-starved parental (Rat1) or dominant-inhibitory Cdc42- (N17Cdc42), Rac1- (N17Rac), or RhoA-expressing (N19Rho) Rat1 fibroblasts were trypsinized, washed, and then replated (more ...)
A Role for Cdc42, Rac, and Rho in the Assembly of Focal Adhesions and Actin Fibers
To determine the roles of Rho family members in integrin-mediated cell morphology, Rat1 cells expressing dominant-inhibitory mutants of Cdc42, Rac, and Rho were plated onto fibronectin for 15 or 60 min and stained for F-actin. At the earliest time point examined (15 min), F-actin–containing ruffles were reduced in the N17Cdc42- and N17Rac-expressing cells (Fig. , e and i) when compared with the Rat1 control cells (Fig. a). In contrast, membrane ruffles were prominent in N19Rho-expressing cells (Fig. m), suggesting that both Cdc42 and Rac (but not Rho) are required for the adhesion-mediated organization of F-actin in membrane ruffles. Subsequent to membrane ruffling, F-actin was organized into bundles. The intensity of phalloidin-stained F-actin in stress fibers appeared to be reduced in the N17Cdc42- (Fig. g) and N17Rac-expressing (Fig. k) cells, and N19Rho expression completely blocked the assembly of these structures (Fig. p). To quantitate the reduction in F-actin observed in Rat1 cells expressing dominant-inhibitory mutants of Cdc42, Rac, or Rho, the cells were plated on fibronectin for 15 or 60 min and the amount of labeled phalloidin bound to F-actin quantitated as described in Materials and Methods. In the cells expressing dominant-inhibitory mutants of Cdc42, Rac, and Rho, F-actin levels were reduced to <50% of the level in control Rat1 cells (Fig. ), suggesting that Cdc42, Rac, and Rho all control the rise in F-actin associated with adherence to fibronectin.
Figure 5 Quantitation of F-actin content in Rat1 cells adhering to fibronectin. Serum-starved parental (Rat1) or dominant-inhibitory Cdc42- (N17Cdc42), Rac1- (N17Rac), or RhoA-expressing (N19Rho) Rat1 fibroblasts were trypsinized, washed, and then replated (more ...)
In parallel with our observations on actin organization, we stained the fibronectin-adherent cells for vinculin-containing focal adhesions. In the N17Cdc42- and N17Rac- expressing cells at the 60-min time point (Fig. , h and l, respectively), the size and/or number of vinculin-containing focal adhesions appeared reduced relative to the Rat1 control cells (Fig. d). In contrast, small focal complexes were observed in the N19Rho-expressing cells at 15 min (Fig. n), but the staining was more dramatically reduced by 60 min so that only a few faint focal adhesions were observed (Fig. q). These results suggest that Rho is essential for adhesion-dependent focal adhesion assembly, but that Cdc42 and Rac also play a role in the assembly of these structures.
To insure that the effects on cell morphology observed in cells expressing N17Cdc42, N17Rac, or N19Rho were not due to an adaptive response of the cells to the low levels of expression of the dominant-inhibitory mutants in uninduced cells, we performed transient transfections of N17Cdc42 or N17Rac1, or treated Rat1 cells with the Rho-specific inhibitor C3 transferase and monitored the adhesion-mediated organization of focal contacts and actin stress fibers. Cells expressing high levels of N17Cdc42 retained a rounded morphology 60 min after being plated onto fibronectin, and these cells had few (if any) detectable actin fibers (Fig. a) or focal contacts (Fig. c). Under the conditions of this experimental system, N17Cdc42 expression had a more pronounced effect on cell morphology (when compared with the stably expressing N17Cdc42 cell lines), perhaps resulting from the higher level of N17Cdc42 expression obtained (Fig. , b and d; and data not shown). Transient expression of N17Rac also inhibited the organization of actin fibers (Fig. e) and focal contacts (Fig. g), similar to what we observed in cells using the tetracycline-repressible system to express N17Rac. C3 transferase inhibition of Rho blocked adhesion-induced stress fiber formation (Fig. i) and focal adhesion organization (Fig. j) in a manner similar to that observed in the N19Rho-expressing Rat1 cells (Fig. , m and n), with only peripheral point contacts and actin fibers detected. These results, obtained in transiently transfected or C3-treated Rat1 cells, serve to support the hypothesis stated above that Cdc42, Rac, and Rho control the assembly of focal adhesions and actin filaments; they suggest that Cdc42 and Rac control the generation of peripheral adhesion/cytoskeletal structures that are generated during cell spreading (an event that may indirectly control stress fiber formation), whereas Rho more directly controls the generation of focal adhesions and actin stress fibers.
Figure 6 The role of Cdc42, Rac, and Rho in the assembly of focal adhesions and stress fibers in Rat1 fibroblasts. Rat1 cells transfected with 10 μg of the pCMV plasmid encoding Myc-N17Cdc42 (a–d) or Myc-N17Rac1 (e–h) or Rat1 cells (more ...)
Adhesion to Fibronectin Induces Integrin-mediated Signals Regulated by Rho and Cdc42
Not only do focal adhesions function as sites where structural components of a cell are assembled to regulate cell morphology, they also serve as a framework for the organization of signaling complexes that control integrin-mediated changes in cell behavior. Since the Rho family of small GTPases regulates several aspects of cell morphology as described above, we set out to identify integrin- mediated signaling events that are Cdc42, Rac, and/or Rho dependent. Since protein phosphorylation is one of the earliest events detected in response to integrin engagement (Guan et al., 1991
; Kornberg et al., 1991
), we chose to examine the role of Rho family GTPases in integrin-mediated tyrosine phosphorylation by observing: (a
) the activation of several kinases (FAK, Src, Erk, and AKT) that are known to be activated by integrin stimulation; and (b
) the tyrosine phosphorylation of some of the potential substrates of these kinases (FAK, Erk, and paxillin).
FAK, a tyrosine kinase that is activated and tyrosine phosphorylated in response to integrin–matrix interactions (Hanks et al., 1992
; Kornberg et al., 1992
; Lipfert et al., 1992
), is also phosphorylated in response to agents that activate Rho (Zachary et al., 1992
; Kumagai et al., 1993
). This makes FAK a likely candidate to be regulated by the Rho family of GTPases in response to adhesion. FAK tyrosine phosphorylation was monitored by immunoblotting FAK immunoprecipitates with a phosphotyrosine antibody (Fig. ). FAK tyrosine phosphorylation in response to plating of Rat1 cells on fibronectin was unaffected in N17Cdc42- or N17Rac-expressing cells (<10%), but N19Rho expression inhibited the tyrosine phosphorylation of FAK by 50% (n
= 5) at the 90-min time point (Fig. A
). Furthermore, when Rat1 cells were treated with C3 transferase to inhibit Rho activity, FAK tyrosine phosphorylation was inhibited by 40–60% (n
= 3) (Fig. C
). Interestingly, we did not observe any inhibition of FAK tyrosine phosphorylation in the N19Rho-expressing cells at the 10-min time point (<5%, n
= 5), suggesting that Rho may not be required for the earliest stages of FAK activation.
Figure 7 A role for Rho in integrin-mediated tyrosine phosphorylation of FAK. In A and B, serum-starved parental (Rat1) or dominant-inhibitory Cdc42- (N17Cdc42), Rac1- (N17Rac), or RhoA-expressing (N19Rho) Rat1 fibroblasts were trypsinized, washed, and then (more ...)
Paxillin is another focal adhesion protein that is tyrosine phosphorylated in response to integrin–matrix interactions (Burridge et al., 1992
). In fact, it is a likely substrate for FAK. Paxillin tyrosine phosphorylation in response to adhesion of Rat1 cells to fibronectin was monitored by anti-phosphotyrosine immunoblotting of paxillin immunoprecipitates. Paxillin tyrosine phosphorylation was reduced by 70% (n
= 3) in the N19Rho-expressing cells at the 90-min time point (Fig. A
) and 50% (n
= 2) in C3 transferase–treated cells (Fig. C
), suggesting that the tyrosine phosphorylation of both FAK and paxillin is at least in part dependent on Rho.
Figure 8 A role for Rho and Cdc42 in integrin-mediated tyrosine phosphorylation of paxillin. In A and B, serum-starved parental (Rat1) or dominant-inhibitory Cdc42- (N17Cdc42), Rac1- (N17Rac), or RhoA-expressing (N19Rho) Rat1 fibroblasts were trypsinized, (more ...)
The mitogen-activated protein (MAP) kinase Erk2 can be activated and phosphorylated downstream of FAK in an integrin-dependent manner (Chen et al., 1994
; Schlaepfer et al., 1994
). Since Erk2 activation is associated with a decrease in the electrophoretic mobility of the protein (or upward shift), we chose to examine the role of Rho family GTPases on Erk2 activity in response to plating of Rat1 cells on fibronectin by observing the mobility shift. Erk2 activation in Rat1 cells was rapid and transient, peaking at ~15 min, and then returning to near basal levels by 90 min (Fig. ). All three dominant-inhibitory mutants (Fig. A
) and C3 transferase treatment (Fig. C
) diminished the activation of Erk2 to varying degrees (N17Cdc42 > C3 > N19Rho > N17Rac), with N17Cdc42 completely inhibiting integrin-mediated Erk2 activation and N17Rac having very little effect. Furthermore, adhesion-induced activation of Erk2 kinase activity, which was enhanced fourfold upon adhesion to fibronectin, was also inhibited when N17Cdc42 was transiently coexpressed (Fig. D
). Interestingly, N17Cdc42 expression did not inhibit Erk2 activation in PDGF-stimulated cells (Fig. B
), suggesting that Cdc42 may strictly be involved in integrin-mediated Erk2 activation.
Figure 9 A role for Rho and Cdc42 in integrin-mediated tyrosine phosphorylation of Erk2. In A, serum-starved parental (Rat1) or dominant-inhibitory Cdc42- (N17Cdc42), Rac1- (N17Rac), or RhoA-expressing (N19Rho) Rat1 fibroblasts were trypsinized, washed, and (more ...)
Src is another protein tyrosine kinase activated upon integrin engagement that may play a role in signaling from the focal adhesion complex (Kaplan et al., 1995
). To examine the role of Rho-family GTPases on Src activity in response to plating cells on fibronectin we immunoblotted detergent lysates with an antibody that specifically recognizes Src when it is phosphorylated at its autophosphorylation site (tyrosine 416) (Gao et al., 1997
). Src was rapidly autophosphorylated in response to plating of Rat1 cells on fibronectin (Fig. ). However, no significant difference (<15%) was observed in Src autophosphorylation in Rat1 cells expressing dominant-inhibitory Rho family mutants, suggesting that integrin-mediated activation of Src is independent of Cdc42, Rac, and Rho.
Figure 10 Integrin-mediated tyrosine phosphorylation of Src is independent of Cdc42, Rac, and Rho. Serum-starved parental (Rat1) or dominant-inhibitory Cdc42- (N17Cdc42), Rac1- (N17Rac), or RhoA-expressing (N19Rho) Rat1 fibroblasts were trypsinized, washed, (more ...)
Akt, an effector for phosphatidylinositol (PI) 3-kinase, is another kinase that can be activated upon attachment to fibronectin (King et al., 1997
). We examined the potential role of Rho family GTPases in integrin-mediated Akt activation by performing immunoprecipitation/kinase assays using histone H2B as an exogenous substrate. Akt activity, which was activated threefold when Rat1 cells were plated on fibronectin, was unaffected by N17Rac or N19Rho expression but inhibited by 55% (n
= 3) in N17Cdc42- expressing Rat1 cells (Fig. A
). The adhesion-induced Cdc42-dependent activation of Akt was further confirmed by transient co-transfection of N17Cdc42 and HA-tagged Akt in Rat1 cells. The twofold activation of Akt, observed after plating these cells on fibronectin for 15 and 60 min, was blocked by N17Cdc42 (Fig. B
). These data suggest that Cdc42 is necessary for integrin-dependent activation of Akt (and, by inference, PI 3-kinase).
Figure 11 Integrin-mediated activation of Akt is Cdc42-dependent. Serum-starved parental (Rat1) or dominant-inhibitory Cdc42- (N17Cdc42), Rac1- (N17Rac), or RhoA-expressing (N19Rho) Rat1 fibroblasts were trypsinized, washed, and then replated on fibronectin-coated (more ...)