Genetic Deletion of Rac1 GTPase Reveals Its Critical Role in Actin Stress Fiber Formation and Focal Adhesion Complex Assembly

University of Cincinnati, Cincinnati, Ohio, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 08/2006; 281(27):18652-9. DOI: 10.1074/jbc.M603508200
Source: PubMed


Rac1 is an intracellular signal transducer regulating a variety of cell functions. Previous studies by overexpression of dominant-negative or constitutively active mutants of Rac1 in clonal cell lines have established that Rac1 plays a key role in actin lamellipodia induction, cell-matrix adhesion, and cell anoikis. In the present studies, we have examined the cellular behaviors of Rac1 gene-targeted primary mouse embryonic fibroblasts (MEFs) after Cre recombinase-mediated deletion of Rac1 gene. Rac1-null MEFs became contracted and elongated in morphology and were defective in lamellipodia formation, cell spreading, cell-fibronectin adhesion, and focal contact formation in response to platelet-derived growth factor or serum. Unexpectedly, deletion of Rac1 also abolished actin stress fibers in the cells without detectable alteration of endogenous RhoA activity. Although the expression and/or activation status of focal adhesion complex components such as Src, FAK, and vinculin were not affected by Rac1 deletion, the number and size of adhesion plaques were significantly reduced, and the molecular complex between Src, FAK, and vinculin was dissembled in Rac1-null cells. Overexpression of an active RhoA mutant or ROK failed to rescue the stress fiber and adhesion plaque defects of the Rac1-null cells. Although Rac1 deletion caused a significant reduction in phospho-PAK1, -AKT, and -ERK under serum stimulation, reconstitution of active PAK1, but not AKT or MEK1, was able to rescue the actin cytoskeleton and adhesion phenotypes of the Rac1-deficient cells. Furthermore, Rac1 deletion led to a marked increase in spontaneous apoptosis that could be rescued by active PAK1, AKT, or MEK1 expression. Our results obtained from gene-targeted primary MEFs indicate that Rac1 is essential not only for lamellipodia induction but also for the RhoA-regulated actin stress fiber and focal adhesion complex formation and that Rac1 is involved in cell survival regulation through anoikis-dependent as well as -independent mechanisms.

20 Reads
  • Source
    • "The same effect has been observed with the inactivation of either NEDD9 or DOCK3, which both mediate the activation of Rac (DOCK3 is a GEF of Rac, NEDD9 is an adaptor protein creating a complex with DOCK3) [37]. Rac1 also influences the assembly of focal adhesions and the subsequent formation of stress fibers [40]. Moreover, Rac suppresses cell contractility through its effector WAVE2 that negatively regulates the phosphorylation of MLC2 and therefore renders the amoeboid mode of motility unfavorable [37]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Tumor cells exhibit at least two distinct modes of migration when invading the 3D environment. A single tumor cell's invasive strategy follows either mesenchymal or amoeboid patterns. Certain cell types can use both modes of invasiveness and undergo transitions between them. This work outlines the signaling pathways involved in mesenchymal and amoeboid types of tumor cell motility and summarizes the molecular mechanisms that are involved in transitions between them. The focus is on the signaling of the Rho family of small GTPases that regulate the cytoskeleton-dependent processes taking place during the cell migration. The multiple interactions among the Rho family of proteins, their regulators and effectors are thought to be the key determinants of the particular type of invasiveness. Mesenchymal and amoeboid invasive strategies display different adhesive and proteolytical interactions with the surrounding matrix and the alterations influencing these interactions can also lead to the transitions.
    Cellular and Molecular Life Sciences CMLS 09/2009; 67(1):63-71. DOI:10.1007/s00018-009-0132-1 · 5.81 Impact Factor
  • Source
    • "The adhesion defects of fibroblasts lacking Rac1 could be partially rescued by transfection with active PAK1 but not active RhoA [25]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Rho family proteins are constitutively activated in the highly invasive human fibrosarcoma HT1080 cells. We now investigated the specific roles of Rac1 and Rac2 in regulating morphology, F-actin organization, adhesion, migration, and chemotaxis of HT1080 cells. Downregulation of Rac1 using specific siRNA probes resulted in cell rounding, markedly decreased spreading, adhesion, and chemotaxis of HT1080 cells. 2D migration on laminin-coated surfaces in contrast was not markedly affected. Selective Rac2 depletion did not affect cell morphology, cell adhesion, and 2D migration, but significantly reduced chemotaxis. Downregulation of both Rac1 and Rac2 resulted in an even more marked reduction, but not complete abolishment, of chemotaxis indicating distinct as well as overlapping roles of both proteins in chemotaxis. Rac1 thus is selectively required for HT1080 cell spreading and adhesion whereas Rac1 and Rac2 are both required for efficient chemotaxis.
    Biochemical and Biophysical Research Communications 07/2009; 386(4):688-92. DOI:10.1016/j.bbrc.2009.06.098 · 2.30 Impact Factor
  • Source
    • "The involvement of Rac3 in cellsubstrate adhesion during cell invasion is a novel finding in this study. Although the molecular mechanism by which Rac regulates cell-substrate adhesion remains unknown, it has been reported that Rac1 depletion suppresses focal complex formation in embryonic fibroblasts and carcinoma cells (Guo et al., 2006; Yip et al., 2007). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The motility of cancer cells in 3D matrices is of two types: mesenchymal motility, in which the cells are elongated and amoeboid motility, in which the cells are round. Amoeboid motility is driven by an actomyosin-based contractile force, which is regulated by the Rho/ROCK pathway. However, the molecular mechanisms underlying the motility of elongated cells remain unknown. Here, we show that the motility of elongated cells is regulated by Rac signaling through the WAVE2/Arp2/3-dependent formation of elongated pseudopodia and cell-substrate adhesion in 3D substrates. The involvement of Rac signaling in cell motility was different in cell lines that displayed an elongated morphology in 3D substrates. In U87MG glioblastoma cells, most of which exhibit mesenchymal motility, inhibition of Rac signaling blocked the invasion of these cells in 3D substrates. In HT1080 fibrosarcoma cells, which display mixed cell motility involving both elongated and rounded cells, inhibition of Rac1 signaling not only blocked mesenchymal motility but also caused a mesenchymal-amoeboid transition. Additionally, Rac1 and RhoA signaling regulated the mesenchymal and amoeboid motility in these cells, respectively, and the inhibition of both pathways dramatically decreased cell invasion. Hence, we could conclude that Rac1 and RhoA signaling simultaneously regulate cell invasion in 3D matrices.
    Oncogene 03/2009; 28(13):1570-83. DOI:10.1038/onc.2009.2 · 8.46 Impact Factor
Show more

Preview (2 Sources)

20 Reads
Available from