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On-Chip Cellomics Assay Enabling Algebraic and Geometric Understanding of Epigenetic Information in Cellular Networks of Living Systems. 1. Temporal Aspects of Epigenetic Information in Bacteria
Sensors 2012, 12(7), 9411-9422; doi:10.3390/s120709411
Article

Dynamics of Ras Complexes Observed in Living Cells

1,2
,
3
 and
1,2,*
1 Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China 2 MoE Key Laboratory for Biomedical Photonics, Department of Biomedical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China 3 Wuhan Mechanical Technology College, Wuhan 430075, China
* Author to whom correspondence should be addressed.
Received: 12 June 2012 / Revised: 29 June 2012 / Accepted: 4 July 2012 / Published: 9 July 2012
(This article belongs to the Special Issue Live Cell-Based Sensors)
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Abstract

K-Ras works as a switch in many important intracellular signaling pathways and plays important roles in cell growth, proliferation, differentiation and carcinogenesis. For signal transduction from K-Ras to Raf1, the best-characterized effector of K-Ras, the general view is that Ras recruits Raf1 from the cytoplasm to the cell membrane. To elucidate this process, we constructed a series of fusion proteins (including Raf1 and K-Ras fused with either fluorescent proteins or fluorescent protein fragments) to compare subcellular localizations of these proteins. Bimolecular fluorescence complementation (BiFC) and a co-transfection system were used. In the BiFC system, the K-Ras/Raf1 complexes were mainly located in the cell membrane, while the Raf1 control was uniformly distributed in the cytoplasm. However, the complexes of Raf1 and K-RasC185S, a K-Ras mutant which loses membrane-localization, were also able to accumulate in the cell membrane. In contrast, an apparent cytosolic distribution pattern was observed in cells co-transfected with mcerulean-Raf1 and EGFP-K-RasC185S, suggesting that the membrane localization of K-Ras/Raf1 complexes is not entirely dependent on K-Ras, and that other factors, such as the irreversible conformation formed between K-Ras and Raf1 may play a role. This study sheds light on the interaction between K-Ras and Raf1 and provides a practical method to elucidate the mechanism underlying K-Ras and Raf1 binding to the cell membrane.
Keywords: K-Ras; Raf1; BiFC; membrane association; signal pathway K-Ras; Raf1; BiFC; membrane association; signal pathway
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).
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Li, X.; Cheng, Z.; Jin, H. Dynamics of Ras Complexes Observed in Living Cells. Sensors 2012, 12, 9411-9422.

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