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

Abstract

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.

Involvement of Rac and Rho signaling in cancer cell motility in 3D substrates

“…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).…”

Combined analysis of Rac1, IQGAP1, Tiam1 and E-cadherin expression in gastric cancer

“…Recent findings indicate that Rac1 may play an important role in the carcinogenesis and progression of gastric carcinoma, because increased Rac1 expression was related to higher TNM stages. 22 Genetic deletion of Rac1 has revealed its critical role in cell survival regulation 23 suggesting that increased expression of Rac1 in tumors may protect cells from apoptosis and lead to an increase of cellmatrix-interaction and cell spread. However, despite these important functions that are attributed to Rac1, no association of expression of Rac1 with prognosis was found in our study of gastric carcinomas.…”

Small GTPase protein Rac-1 is activated with maturation and regulates cell morphology and function in chondrocytes

“…DN-Rac-1 and -Cdc42 expression inhibit cell spreading in NIH3T3 cells on several substrates [38] and Rac-1 deficiency in fibroblasts causes cell rounding on fibronectin. [19,27] Further, Rac-1 activation in endothelial cells induces spreading, even in integrin kinase deficient cells. [39] RhoA, as well as Rac-1, has strong effects on the reorganization of cytoskeletal structures and thus, directs changes in cell shape in various cell types that grow in an anchorage dependent manner.…”

“…[20,27] Thus, we first determined the ability of Rac-1 to modulate cell attachment and spreading. The infected chondrocytes were plated on collagen type I, fibronectin, gelatin, or collagen type II substrates and cell attachment was quantified by computer-assisted analysis one hour after plating.…”