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Inactivation of the small GTPase Rho disrupts cellular attachment and induces adhesion-dependent and
D Bobak1, J Moorman, A Guanzon
1Department of Medicine, Beirne Carter Center for Immunology Research, University of Virginia School of Medicine, Charlottesville 22908, USA.
Abstract:
Rho small GTPases regulate a variety of cellular signaling pathways involved in cell growth and transformation. In this study, we examined potential roles for Rho in adhesion-dependent and -independent pathways regulating apoptosis. Rho GTPases are specifically inactivated by exoenzyme C3 (C3) of Clostridium botulinum. Using a novel Sindbis virus-based gene expression system, we created a double subgenomic recombinant (dsSIN:C3) capable of expressing active C3 in intact cells. Infection of L929 fibroblasts with dsSIN:C3 caused essentially complete ADP-ribosylation of intracellular Rho within 1 h. dsSIN:C3-infected cells also became rounded within 1-2 h and detached by 5 h post-infection. Infection of L929 in suspension with dsSIN:C3 disrupted the ability for normal cellular attachment and spreading. Infection of primary cell explants of chicken embryo fibroblasts (CEF) and rat aortic smooth muscle cells (RSM) with dsSIN:C3 caused cytoskeletal effects similar to those seen in L929. We also observed that C3 markedly decreased the basal phosphorylation state of focal adhesion kinase (FAK). Most intriguingly, we found that dsSIN-based expression of C3 or loss of function mutants of Rho could each induce apoptosis and, in RSM, this effect was observed to be adhesion-independent. Rho GTPases, therefore, appear to regulate signal pathways that are required for cell survival and growth that are separate from, but likely overlap with, Rho-dependent pathways involved in cellular adhesion.
Insights
Rho GTPases are crucial for cell survival and growth, regulating pathways distinct from those controlling cell adhesion. Inhibiting Rho GTPases with Clostridium botulinum exoenzyme C3 induces apoptosis, highlighting their role in preventing programmed cell death.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Rho GTPases are key regulators of cellular signaling pathways governing cell growth, transformation, and adhesion.
- Understanding the role of Rho GTPases in apoptosis is crucial for comprehending cell survival and death mechanisms.
Purpose of the Study:
- To investigate the role of Rho GTPases in both adhesion-dependent and -independent pathways regulating apoptosis.
- To explore the effects of Rho GTPase inactivation on cellular morphology, attachment, and survival.
Main Methods:
- Utilized a Sindbis virus-based gene expression system (dsSIN:C3) to deliver active exoenzyme C3 (C3) from Clostridium botulinum into intact cells.
- Infected various cell types, including L929 fibroblasts, chicken embryo fibroblasts (CEF), and rat aortic smooth muscle cells (RSM), with dsSIN:C3.
- Assessed Rho GTPase activity via ADP-ribosylation, monitored cellular morphology and attachment, and analyzed the phosphorylation state of focal adhesion kinase (FAK).
Main Results:
- Infection with dsSIN:C3 led to rapid and near-complete inactivation of intracellular Rho GTPases.
- dsSIN:C3-infected cells exhibited characteristic rounding, detachment, and disrupted cellular attachment and spreading.
- C3 treatment reduced focal adhesion kinase (FAK) phosphorylation and, significantly, induced apoptosis in a manner that was adhesion-independent in rat aortic smooth muscle cells.
- Expression of C3 or Rho loss-of-function mutants triggered apoptosis, indicating a pro-survival role for Rho GTPases.
Conclusions:
- Rho GTPases play a critical role in regulating cell survival and growth through pathways that are distinct from, yet likely overlap with, Rho-dependent adhesion pathways.
- Inactivation of Rho GTPases can lead to apoptosis, suggesting these proteins are essential for preventing programmed cell death.
- The findings reveal novel, non-canonical functions of Rho GTPases in maintaining cell viability.