This journal is c The Royal Society of Chemistry 2012 Mol. BioSyst. Cite this: DOI: 10.1039/c2mb25100k Robustness analysis of cellular systems using the genetic tug-of-war methodwz Hisao Moriya,* a Koji Makanae, a Kenji Watanabe, b Ayako Chino ac and Yuki Shimizu-Yoshida d Received 16th March 2012, Accepted 14th May 2012 DOI: 10.1039/c2mb25100k Robustness is one of the principles of design inherent to biological systems. Cellular robustness can be measured as limits of intracellular parameters such as gene expression levels. We have recently developed an experimental approach coined as genetic Tug-Of-War (gTOW), which we used to perform robustness analysis in yeast. Using gTOW, we were able to measure the upper limit of expression of gene targets. In this review, we first elaborate on how the gTOW method compares to current mathematical simulation models prevalently used in the determination of robustness. We then explain the experimental principles underlying gTOW and its associated tools, and we provide concrete examples of robustness analysis using gTOW, i.e. cell cycle and HOG pathway gene expression analysis. Finally, we list a series of Q&As related to the experimental utilization of gTOW and we describe the potential impact of gTOW and its relevance to the understanding of biological systems. Introduction The capture of life as biological systems became possible thanks to the progress made in molecular biology during the 20th century. 1 Robustness, defined as the ability of a system to maintain its function despite perturbations, is commonly observed within biological systems, and has now evolved into one of the central themes in systems biology. 2,3 For example, cellular systems involved in bacterial chemotaxis as well as body development patterns in the Drosophilia embryo are known to be robust against the fluctuations of intracellular protein levels. 4,5 In this review, we will first introduce how cellular robustness may be assessed as limits of intra- cellular parameters using mathematical simulation models, then we will elaborate on the principles and tools associated with the recently developed genetic tug-of-war (gTOW) method, which aims to carry out robustness analysis experimentally. In a final section, we explain the scientific knowledge that may be revealed from robustness analysis using the gTOW approach. Robustness itself is a complicated concept. 6–9 The scope of this paper is not reviewing the theoretical studies of robustness, but explaining the current progress in robustness measurements in vivo using the gTOW method. Robustness of simple cell cycle models To help understand the robustness of cellular systems against fluctuations of intracellular parameters, we explain computer simulations using simplified mathematical models of the cell a Research Core for Interdisciplinary Sciences, Okayama University, Japan. E-mail: hisaom@cc.okayama-u.ac.jp b School and Graduate School of Pharmaceutical Sciences, University of Shizuoka, Japan c Graduate School of Natural Science and Technology, Okayama University, Japan d Sony Computer Science Laboratory, Incorporated, Japan w Published as part of a themed issue dedicated to Emerging Investigators. z Electronic supplementary information (ESI) available. See DOI: 10.1039/c2mb25100k Hisao Moriya Hisao Moriya is a designated Associate Professor of Research Core for Interdisciplinary Sciences, Okayama University. He completed his PhD in Biology from Kobe University in 1998 under the supervision of Professor Katsumi Isono. After his engagement in the study of the glucose sensing system in yeast at Prof. Mark Johnston’s lab in Washington University, he joined to Dr Hiroaki Kitano’s group (Kitano Symbiotic Systems Project, JST), where he started the study on robustness of cellular systems which is the main topic of his current research. In 2006, he was selected as a researcher of JST PRESTO project ‘‘The Dynamic Mechanism of and Fundamental Technology for Biological System’’. In 2009, he moved to Okayama University where he started his own group. Molecular BioSystems Dynamic Article Links www.rsc.org/molecularbiosystems REVIEW Downloaded by Okayama Daigaku on 29 June 2012 Published on 21 June 2012 on http://pubs.rsc.org | doi:10.1039/C2MB25100K View Online / Journal Homepage