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Updated: Mar 31, 2026
Quantitative Analysis of Cell Edge Dynamics during Cell Spreading
Published on: May 22, 2021
RLIP76 regulates Arf6-dependent cell spreading and migration by linking ARNO with activated R-Ras at recycling
Jeremy G T Wurtzel1, Seunghyung Lee1, Sharad S Singhal2
1Department of Anatomy & Cell Biology and The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA, United States.
Abstract:
R-Ras small GTPase enhances cell spreading and motility via RalBP1/RLIP76, an R-Ras effector that links GTP-R-Ras to activation of Arf6 and Rac1 GTPases. Here, we report that RLIP76 performs these functions by binding cytohesin-2/ARNO, an Arf GTPase guanine exchange factor, and connecting it to R-Ras at recycling endosomes. RLIP76 formed a complex with R-Ras and ARNO by binding ARNO via its N-terminus (residues 1-180) and R-Ras via residues 180-192. This complex was present in Rab11-positive recycling endosomes and the presence of ARNO in recycling endosomes required RLIP76, and was not supported by RLIP76(Δ1-180) or RLIP76(Δ180-192). Spreading and migration required RLIP76(1-180), and RLIP76(Δ1-180) blocked ARNO recruitment to recycling endosomes, and spreading. Arf6 activation with an ArfGAP inhibitor overcame the spreading defects in RLIP76-depleted cells or cells expressing RLIP76(Δ1-180). Similarly, RLIP76(Δ1-180) or RLIP76(Δ180-192) suppressed Arf6 activation. Together these results demonstrate that RLIP76 acts as a scaffold at recycling endosomes by binding activated R-Ras, recruiting ARNO to activate Arf6, thereby contributing to cell spreading and migration.
Insights
RLIP76 acts as a scaffold protein, linking R-Ras to ARNO at recycling endosomes to activate Arf6. This process is crucial for cell spreading and motility.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- R-Ras small GTPase regulates cell spreading and motility.
- RalBP1/RLIP76 is an R-Ras effector involved in activating Arf6 and Rac1 GTPases.
Purpose of the Study:
- To elucidate the mechanism by which RLIP76 enhances cell spreading and motility.
- To investigate the role of RLIP76 in connecting R-Ras to Arf6 activation at recycling endosomes.
Main Methods:
- Co-immunoprecipitation to detect protein complexes.
- Recycling endosome localization studies using Rab11 marker.
- Analysis of cell spreading and migration assays.
- Expression of RLIP76 deletion mutants.
Main Results:
- RLIP76 forms a complex with R-Ras and ARNO (cytohesin-2) at recycling endosomes.
- RLIP76's N-terminus (1-180) binds ARNO, while residues 180-192 bind R-Ras.
- RLIP76 is essential for ARNO recruitment to recycling endosomes and subsequent Arf6 activation.
- RLIP76 deletion mutants impair Arf6 activation and cell spreading/migration.
Conclusions:
- RLIP76 functions as a scaffold protein at recycling endosomes.
- RLIP76 bridges activated R-Ras and ARNO to facilitate Arf6 activation.
- This R-Ras-RLIP76-ARNO-Arf6 pathway is critical for cell spreading and migration.
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