ANALYTICAL SCIENCES NOVEMBER 2016, VOL. 32 1151 2016 © The Japan Society for Analytical Chemistry Reviews Integrin-independent Cell Adhesion Substrates: Possibility of Applications for Mechanobiology Research Takashi HOSHIBA*,**† and Masaru TANAKA*,*** * Frontier Center for Organic Materials, Yamagata University, 4-3-16 Jonan, Yonezawa, Yamagata 992–8510, Japan ** International Center for Materials Nanoarchitectonics, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305–0044, Japan *** Institute for Materials Chemistry and Engineering, Kyushu University, 744 Motooka, Nishi, Fukuoka, Fukuoka 819–0395, Japan Cells can mainly sense mechanical cues from the extracellular matrix via integrins. Because mechanical cues can strongly influence cellular functions, understanding the roles of integrins in the sensing of mechanical cues is a key for the achievement of tissue engineering. The analyses to determine the roles of integrins in the sensing of mechanical cues have been performed by many methods based on molecular- and cell-biological techniques, atomic force microscopy, and optical tweezers. Integrin-dependent cell adhesion substrates have been also used for this purpose. Additionally, the cells can adhere on several substrates via integrin-independent mechanisms. There are two types of integrin-independent cell adhesion substrates; 1) the substrates immobilized with ligands against the receptors on cell surface and 2) the substrates suppressing protein adsorption. Cells can exhibit specific functions on these substrates. Here, the examples of integrin- independent cell adhesion substrates were reviewed, and their possible applications in mechanobiology research are discussed. Keywords Integrin, cell adhesion, extracellular matrix, receptor, protein adsorption, signal transduction (Received June 30, 2016; Accepted September 1, 2016; Published November 10, 2016) 1 Introduction 1152 substrate surface 2 The Substrates that Promote 3·4 A comparison of the two types of Integrin-dependent Adhesion 1153 integrin-independent cell-adhesive substrates 3 The Substrates that Allow 3·5 Perspectives on the application of Integrin-independent Cell Adhesion 1153 integrin-independent cell adhesion substrates 3·1 The importance of the substrates used for for mechanobiology research integrin-independent cell adhesion 4 Conclusions 1157 3·2 The substrate-immobilization of ligands against 5 Acknowledgements 1157 non-integrin receptors on the cell membrane 6 References 1157 3·3 Suppression of protein adsorption onto the Takashi HOSHIBA received his Ph.D. Masaru TANAKA is a professor at Kyushu degree in 2007 from Tokyo Institute of University. He worked for TERUMO Co. Technology. He worked at National and designed novel biocompatible Institute for Materials Sciences (NIMS) polymers and commercialized (World as a PostDoc fellow (2007 – 2009) and as shear No. 1). In 2000 he moved to a Research Fellow of the Japan Society Hokkaido University and became a JST for Promotion of Science (2009 – 2011). PRESTO researcher, and then moved to He has been in Yamagata University as a Tohoku University. Medical devices Research Assistant Profesor (2011 – 2014) covered with the 3D films are and also been as a Research Associate commercially available in the world Professor (2014 –). Also, he has been as a clinical market. In 2009, he has full Guest Researcher in NIMS (2013 –). His professorship at Yamagata University. He current research interests are extracellular became a leader of Funding Program for matrix and biomaterials. Next Generation World-Leading Researchers (2011 – 2014). He has received the Awards including SPSJ Asahi Kasei Award for the intermediate † To whom correspondence should be addressed. water concept based on the role of interfacial water at the materials E-mail: [email protected] interphases. 1152 ANALYTICAL SCIENCES NOVEMBER 2016, VOL. 32 To understand the mechanisms of cell function regulation by 1 Introduction biochemical cues, the mechanisms by which integrins and intracellular signaling proteins at the integrin-ECM adhesion The extracellular microenvironment, particularly the sites transduce the biochemical cues provided by the ECM into extracellular matrix (ECM), is one of the most important points the cells have been actively examined.13,23–25 There are several of study in the fields of tissue engineering and regenerative methods for modulating integrin-dependent cell adhesion that medicine because cells can sense information contained in ECM have been used to determine these mechanisms. Molecular- and and modulate their functions in cell proliferation, migration, cell biology-based methods have been frequently used for this morphogenesis and differentiation.1,2 There have been many purpose (Table 1). For example, integrin-dependent cell efforts to regulate cell function by biochemical cues in the past adhesion can be promoted by the induction of integrin over- few decades. ECM proteins and cell-adhesive peptides (e.g., the expression. On the other hand, integrin-dependent cell adhesion Arg-Gly-Asp peptide) have been frequently immobilized to cell can be inhibited by the knock-out or knock-down of integrins culture substrates to provide biochemical cues to the cells.3–6 In and by the use of function-neutralizing antibodies against addition to biochemical cues, the ECM can provide mechanical integrins.6,26–28 In addition to these molecular- and cell biology- cues to the cells to regulate cell function.7–9 Durotaxis is one of based methods, integrin-specific ligands immobilized to the the first phenomena to be reported as a cell function regulated substrates have also been frequently used to promote integrin- by mechanical cues from the ECM.7 Durotaxis is a mode of cell dependent cell adhesion. These substrates have been applied for migration regulated by the elasticity of the ECM; that is, most the analyses of the roles of integrins in the transduction of normal cells prefer to migrate onto stiffer ECM. More biochemical cues provided by the ECM (Table 1).13,23,29 There importantly, the differentiation of stem cells is strongly are also several substrates that allow integrin-independent cell influenced by the stiffness of the ECM.8 Therefore, mechanical adhesion. These approaches that use substrates have also been cues have been a focus of studies in tissue engineering and used for the analyses of the roles of integrins in the transduction regenerative medicine. of biochemical cues provided by the ECM (Table 1).30,31 It has been reported that cell adhesion sites to ECM are highly In contrast to the analyses of the roles of integrins in the important for sensing the biochemical cues provided by the transduction of biochemical cues, the methods are limited for ECM. Cells mainly adhere to the ECM via receptor proteins on the analyses of the roles of integrins in the transduction of the cell membrane called integrins. Integrins are heterodimers mechanical cues provided by the ECM. For this purpose, the composed of α and β transmembrane subunits that have analyses have been performed using molecular- and the cell extracellular domains that can bind to many ECM proteins.10 biology-based methods (e.g. genetic mutation technique and The binding of integrins to ECM proteins leads to the clustering Förster resonance energy transfer (FRET)-based molecular of integrins on the cell membrane. This clustering leads to the tension sensors)32,33 and mechanical cell manipulation methods accumulation of many intracellular proteins (e.g., focal adhesion (e.g. cell manipulations with atomic force microscope and kinase (FAK), talin, paxillin, vinculin and p130Cas) and actin optical tweezer techniques).34,35 In addition to these approaches, fibers close to the intracellular domains of integrins for the the substrates were developed to measure traction forces exerted transduction of biochemical cues provided by the ECM.10–12 by the cells. Microbeads embedded elastic substrates have been There are many types of α and β subunit associations which can used to measure the force exerted by the cells to the substrates, activate different intracellular signaling pathways and induce which is called traction force microscopy.36 Additionally, the different cell functions.10,13 substrates possessing micropillars on the surface have been also In addition to biochemical cues, mechanical cues from ECM used to measure the force exerted by the cells.37 These substrates are also sensed by integrins and intracellular signaling proteins allow the cells to interact and adhere through integrin-dependent at integrin-ECM adhesion sites although mechanical cues from mechanism. The substrates that allow integrin-independent cell extracellular milieu can be sensed by many receptors such as adhesion have been hardly used. Using substrates that allow cadherin and ion channels.14–17 The cells adhere to the substrates integrin-independent cell adhesion might pave the way toward and exert traction forces. When the cells adhere to substrates developing a new method for determining how integrins with different stiffnesses, they exert traction forces with different transduce mechanical cues from the ECM. In this review, we strengths.18 The exertion of traction forces with different summarize examples of the substrates that allow integrin- strengths leads to changes in the conformation, accumulation independent cell adhesion after a brief discussion of the and phosphorylation levels of intracellular signaling proteins at substrates that promote integrin-dependent