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Requirement for C3G-dependent Rap1 activation for cell adhesion and embryogenesis
1Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan.
Abstract:
C3G is a guanine nucleotide exchange factor (GEF) for Rap1, and is activated via Crk adaptor protein. To understand the physiological role of C3G, we generated C3G knockout mice. C3G(-/-) homozygous mice died before embryonic day 7.5. The lethality was rescued by the expression of the human C3G transgene, which could be excised upon the expression of Cre recombinase. From the embryo of this mouse, we prepared fibroblast cell lines, MEF-hC3G. Expression of Cre abolished the expression of C3G in MEF-hC3G and inhibited cell adhesion-induced activation of Rap1. The Cre-expressing MEF-hC3G showed impaired cell adhesion, delayed cell spreading and accelerated cell migration. The accelerated cell migration was suppressed by the expression of active Rap1, Rap2 and R-Ras. Expression of Epac and CalDAG-GEFI, GEFs for Rap1, also suppressed the accelerated migration of the C3G-deficient cells. This observation indicated that Rap1 activation was sufficient to complement the C3G deficiency. In conclusion, C3G-dependent activation of Rap1 is required for adhesion and spreading of embryonic fibroblasts and for the early embryogenesis of the mouse.
Insights
C3G guanine nucleotide exchange factor (GEF) is essential for early mouse development. Its absence causes embryonic lethality, impacting cell adhesion, spreading, and migration by disrupting Rap1 activation.
Area of Science:
- Cell Biology
- Developmental Biology
- Molecular Biology
Background:
- C3G functions as a guanine nucleotide exchange factor (GEF) for Rap1.
- C3G activation is mediated by the Crk adaptor protein.
- The physiological role of C3G in mammalian development is largely unknown.
Purpose of the Study:
- To investigate the essential role of C3G in early mouse embryogenesis.
- To elucidate the function of C3G in regulating Rap1 activation and cellular processes.
Main Methods:
- Generation of C3G knockout mice.
- Rescue of embryonic lethality using a human C3G transgene.
- Establishment of C3G-deficient fibroblast cell lines (MEF-hC3G) via Cre-mediated excision.
- Analysis of cell adhesion, spreading, and migration dynamics.
Main Results:
- C3G knockout mice exhibit embryonic lethality before day 7.5.
- C3G deficiency in MEF-hC3G cells impairs cell adhesion and spreading, while accelerating cell migration.
- Rap1 activation is crucial for complementing C3G deficiency, as evidenced by suppressed accelerated migration with active Rap1, Rap2, R-Ras, Epac, and CalDAG-GEFI expression.
Conclusions:
- C3G-dependent Rap1 activation is indispensable for embryonic fibroblast adhesion and spreading.
- C3G plays a critical role in early mouse embryogenesis, likely through its regulation of Rap1 signaling.