In this study we have performed a comprehensive comparison of the role of individual members of the entire family of Cdc42/Rac GTPases in PDGF-dependent chemotaxis of primary fibroblasts using a single, well-defined cell assay. In addition, the combined knockdown of multiple family members has provided insights into the cooperative roles of these GTPases during cell migration. Using our experimental system, we have demonstrated roles for Cdc42, Rac1, and RhoG in fibroblasts chemotaxis, while other related GTPases, including Tc10, Tcl, Wrch1, Rac2, and Rac3, were all found to be dispensable for this process. Findings from our study suggest that during fibroblast migration partial redundancy exists among Cdc42 and Rac family GTPases. In the absence of Cdc42, Rac1, or RhoG, cell migration can still occur, albeit less efficiently, as cells exploit alternative modes of movement. In the absence of Cdc42, cells adopt a retracted morphology, and migration is accompanied by the extension of narrow processes, while in the absence of either Rac1 or RhoG cell migration is largely accompanied by the extension of filopodia and other thicker membrane extensions.
Cdc42, Rac1, and RhoG may therefore contribute to different aspects of cell migration, and it may therefore be the case that, following the loss of one of these GTPases, the cell adopts a mode of migration that exploits the functions of the remaining proteins. However, when either Cdc42 and Rac1 or Cdc42, Rac1, and RhoG levels are suppressed simultaneously, cell migration is severely inhibited, since the capacity for cells to form dynamic actin-based protrusive structures is greatly impaired. Interestingly, despite the severe effects that combined depletion of Cdc42, Rac1, and RhoG had on the migration of primary fibroblasts, residual cell movement was directed toward the PDGF source. These findings demonstrate that Cdc42/Rac family GTPases regulate migration speed but not the directional response to the gradient during chemotaxis.
In our study we demonstrate that Cdc42 knockdown in MEFs results in impaired chemotaxis. However, Cdc42 knockdown cells can detect and migrate toward the increasing concentration of PDGF, and the resultant mean direction of cell migration is no different than that of the control cells from the same cultures. We show that reduced speed is the major factor contributing to impaired chemotaxis in Cdc42 knockdown cells and demonstrate that while defects in the persistence of these cells do exist, this does not significantly impair their directional response to the PDGF gradient. Yang et al. report reduced migration of Cdc42-null fibroblasts in a serum gradient using a transwell assay and therefore concluded that directionality is impaired in these cells. The difference in their findings and the findings from the present study may reflect differences in the experimental assays used or the chemotactic stimulus used. The gradient characteristics for a given chemoattractant in terms of stability, steepness, and half-life are likely to differ for the transwell chamber and the Dunn chamber. Therefore, we cannot discount the possibility that Cdc42 knockdown may result in defects in the directional response when the gradient characteristics of PDGF differ from those in our study (for example, in shallow gradients of PDGF). However, given that Yang et al., like us, also report reduced cell migration speed after inhibition of Cdc42 signaling, impaired migration speed alone can account for the reduced transwell migration observed in their study. By direct observation of cell migration and subsequent careful analysis of cell trajectories, we gain detailed insights into the various aspects of migration contributing to the chemotactic response of these cells.
The reduced speed of fibroblast migration after the loss of Cdc42 function reported by us and others contrasts with findings from cells of hematopoietic origin, where deficiencies in Cdc42 signaling have actually been found to enhance migration speed (2
). A study using the Bac1 macrophage cell line first ascribed a role for Cdc42 in chemotaxis by demonstrating that microinjection of dominant-negative Cdc42 into macrophages disrupts chemotaxis toward CSF-1, while slightly enhancing migration speed (2
). A study of Drosophila
hemocyte migration in vivo demonstrates that loss of Cdc42 function in these cells results in reduced directional persistence but also significantly enhanced migration speeds (24
). Indeed, the authors of that study demonstrated that while Cdc42-null hemocytes follow a more torturous path to their destination, enhanced migration speed allows them to reach their target as quickly as wild-type cells. It is therefore clear that while Cdc42 plays an important role in cell migration in both fibroblastlike cells and cells of hematopoietic origin, clear differences exist in its regulation of various aspects of this behavior. A phenotype associated with impaired Cdc42 signaling that does, however, appear to be common to many cell types is retraction and elongation of the cell body. It is interesting, therefore, that we have found that basal levels of Rho activity are reduced in Cdc42 knockdown fibroblasts. Given the similarity between the Cdc42 knockdown phenotype and the C3 phenotype observed in our cells, it is tempting to postulate that reduced Rho activity may contribute to the cell retraction observed in cells deficient for Cdc42 signaling.
In the present study we also demonstrate that Rac1 and RhoG, but not Rac3, regulate the morphology and migration of primary fibroblasts. The direct observation of Rac1 and RhoG knockdown cells in a PDGF-BB gradient demonstrates that, in the absence lamellipodia, cells migrate primarily via the extension of filopodia and other thicker membrane processes and that the directional response to the gradient of cells utilizing this mode of migration is as efficient as that observed for cells under control conditions where broad lamellipodia are the dominant protrusive structures. The chemotactic ability of Rac1-deficient cells is consistent with two recent studies characterizing the motility of Rac1-null (28
) and Rac1 knockdown (14
) fibroblasts, in which transwell assays were used to demonstrate the chemotaxis of Rac1-deficient fibroblasts toward PDGF-BB and fMLP, respectively.
The morphological phenotypes observed after knockdown of Rac1 and Cdc42 in MEF is consistent with the previous report by Srinivasan et al. (23
), who examined the roles of these GTPases in neutrophil migration and chemotaxis. These authors demonstrated distinct roles for Rac1 and Cdc42 in the regulation of lamellipodia during neutrophil migration, with the former controlling the formation of these structures and the latter their stability. These authors further demonstrated the loss of chemotaxis toward fMLP in neutrophils expressing a dominant-negative Cdc42 construct. The fact that we still observed migration toward PDGF in MEFs after knockdown of Cdc42 could either reflect differences in the cell types studied or differences in the signaling pathways used during the chemotactic response to these different signaling molecules. Since we were unable to detect significant MEF chemotaxis toward fMLP using our experimental system, this latter possibility could not be addressed.
Given that Rac1 is required for efficient chemotaxis of MEFs in a PDGF gradient, it is interesting that PDGF stimulation only results in a brief, transient burst of Rac1 activity, with active Rac1 returning to a level comparable to that found for the resting state thereafter. It is possible that the initial and transient burst of Rac1 activity functions to mobilize the actin polymerizing machinery in order to enable the cell to make the transition from a quiescent state to a motile state in response to the PDGF stimulus. Once the cell has adopted the motile state, Rac1 would need to be downregulated to prevent excessive activation, which may have deleterious effects for cell motility and polarity, with local changes in the levels of active Rac1 and Cdc42 then maintaining the steady turnover of the actin cytoskeleton during cell migration.
RhoG has previously been implicated in both fibroblast migration and the regulation of Rac1 activity and, consequently, it has been suggested that RhoG regulates cell motility through the downstream regulation of Rac1 (10
). However, in the present study we have shown that, although RhoG is also required for efficient cell migration, inhibition of RhoG expression has no effect either on the basal level of Rac1 activity or on the PDGF-dependent activation of Rac1. Furthermore, combined knockdown of both RhoG and Rac1 inhibited the speed of cell migration to a greater extent than that found for knockdown of Rac1 alone. Although RhoG knockdown does not influence the activity of Rac1, these findings do not necessarily exclude a role for RhoG in the regulation of Rac1-mediated cell migration. For example, the loss of RhoG may influence the efficiency of Rac1 function by removing a subset of Rac1 activators critical for migration or by mislocalizing adaptor molecules critical for the efficient recruitment of active Rac1 to specific subcellular compartments during migration. However, findings from the present study do suggest that RhoG can contribute to cell migration through mechanisms other than those involved in the regulation of Rac1.
Finally, our recombinant C3 microinjection studies clearly demonstrate that under conditions where Rho activity is highly attenuated, fibroblasts can chemotax efficiently toward PDGF. Interestingly, Nobes and Hall (13
) have previously reported that while microinjection of recombinant, dominant-active RhoA has a severe inhibitory effect on primary rat fibroblast migration during a scratch wound assay, microinjection of C3 has a far less pronounced effect on cell migration despite having profound effects on cell morphology. Clear inhibition of wound closure could only be observed in their study when C3 was microinjected at a concentration well above that required to severely alter cell morphology and abolish stress fibers. Under such conditions, cells often lost attachment to the substrate, indicating the essential role of Rho in maintaining cell adhesion. It has recently been shown that ROCK-1-deficient mice exhibit defective closure of both the eye lids and the ventral body wall during embryonic development (22
). In these mice, epithelial cells at the leading margin of the eyelids are unable to organize actomyosin bundles, which appear to be required for the efficient migration of these cells as a sheet across the underlying eye. It may therefore be the case that Rho signaling is more critical for the coordinated migration of a cell population as a single sheet or monolayer, where a “purse string”-based mechanism utilizing actomyosin machinery provides contractile forces that aid the process of forward migration. Our findings suggest, however, that the migration of isolated cells in response to a chemotactic signal is less dependent on Rho function, where the purse-string mechanism of migration does not apply.
In summary, we have performed a comprehensive and detailed analysis of the role of specific Cdc42/Rac family GTPases in the chemotaxis of primary fibroblasts. Our study demonstrates that Cdc42, Rac1, and RhoG function cooperatively during cell migration and reveals new insights into the relationship of these GTPases in the regulation of cell movement. We also demonstrate that while each of these GTPases is implicated in the control of cell morphology and speed, these and other Cdc42/Rac-related GTPases are not required for the directional response toward PDGF during the chemotaxis of primary fibroblasts. Our experimental approach, which combines the Dunn chamber with fluorescent cell labeling techniques, siRNA-mediate protein knockdown, and time-lapse microscopy, provides an extremely powerful method for assessing the significance of differences observed between different treatments, since direct comparisons can be made between control and test cell populations within the same chemotaxis experiment. We believe that this assay will be useful for the future study of various signaling molecules in the migration and chemotaxis of cells in vitro.