Integrin-Specific Mechanoresponses to Compression and Extension Probed by Cylindrical Flat-Ended AFM Tips in Lung Cells Irene Acerbi1,2,3, Toma´ s Luque1,3, Alı´cia Gime´ nez1, Marta Puig1,4, Noemi Reguart5, Ramon Farre´ 1,4,5, Daniel Navajas1,3,5, Jordi Alcaraz1,5* 1 Unitat de Biof´ısica i Bioenginyeria, Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain, 2 Laboratorio di Tecnologie Biomediche, Dipartimento di Bioingegneria, Politecnico di Milano, Milano, Italy, 3 Institut de Bioenginyeria de Catalunya (IBEC), Barcelona, Spain, 4 Institut d’Investigacions Biome`diques August Pi i Sunyer (IDIBAPS), Barcelona, Spain, 5 CIBER de Enfermedades Respiratorias (CIBERES), Bunyola, Spain Abstract Cells from lung and other tissues are subjected to forces of opposing directions that are largely transmitted through integrin-mediated adhesions. How cells respond to force bidirectionality remains ill defined. To address this question, we nanofabricated flat-ended cylindrical Atomic Force Microscopy (AFM) tips with ,1 mm2 cross-section area. Tips were uncoated or coated with either integrin-specific (RGD) or non-specific (RGE/BSA) molecules, brought into contact with lung epithelial cells or fibroblasts for 30 s to form focal adhesion precursors, and used to probe cell resistance to deformation in compression and extension. We found that cell resistance to compression was globally higher than to extension regardless of the tip coating. In contrast, both tip-cell adhesion strength and resistance to compression and extension were the highest when probed at integrin-specific adhesions. These integrin-specific mechanoresponses required an intact actin cytoskeleton, and were dependent on tyrosine phosphatases and Ca2+ signaling. Cell asymmetric mechanoresponse to compression and extension remained after 5 minutes of tip-cell adhesion, revealing that asymmetric resistance to force directionality is an intrinsic property of lung cells, as in most soft tissues. Our findings provide new insights on how lung cells probe the mechanochemical properties of the microenvironment, an important process for migration, repair and tissue homeostasis. Citation: Acerbi I, Luque T, Gime´nez A, Puig M, Reguart N, et al. (2013) Integrin-Specific Mechanoresponses to Compression and Extension Probed by Cylindrical Flat-Ended AFM Tips in Lung Cells. PLoS ONE 7(2): e32261. doi:10.1371/journal.pone.0032261 Editor: Pere-Joan Cardona, Fundacio´ Institut d’Investigacio´ en Cie`ncies de la Salut Germans Trias i Pujol - Universitat Auto` noma de Barcelona - CIBERES, Spain Received September 6, 2011; Accepted January 24, 2012; Published February 23, 2012 Copyright: 2012 Acerbi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Ministerio de Ciencia e Innovacio´ n (SAF2009-13243 to JA, SAF2008-02991 to RF www.micinn.es), Ministerio de Sanidad y Pol´ıtica Social (PI081908 to DN, PS09/01377 to NR www.msps.es), Asociacio´ n Espan˜ ola Contra el Ca´ncer (to NR and JA www.aecc.es), and pre-doctoral fellowships from the Ministerio de Ciencia e Innovacio´ n (to IA, TL and AG), and from the Fundaci o´ Privada Cellex (to MP). No additional external funding was received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jalcaraz@ub.edu Introduction Tissue cells are subjected to a variety of dynamic mechanical stimuli during physiological processes including development, normal organ function, and in a long list of diseased conditions [1,2]. The presence of dynamic mechanical stimuli is particularly obvious in the lung, where cells from both the parenchymal and the stromal compartments continuously experience cyclic stretch- ing forces due to breathing [1,3]. At the cellular level, a major effect of dynamic stretch is that cells experience forces of opposing directions (i.e. bidirectional), such as compression and extension. The normal cell mechanical response to these bidirectional forces is essential for normal lung function. Conversely, a hallmark of prevalent respiratory diseases including asthma and fibrosis is an abnormal mechanical behavior of lung cells, concomitant with an impairment of lung functions [1,3]. Rather than acting globally on tissue cells, mechanical forces are ultimately conveyed to cells locally through their adhesion sites to neighboring cells or to their surrounding extracellular matrix (ECM) [4,5,6]. Previous studies have highlighted the prominent role of the integrin family of transmembrane ECM receptors in conveying extracellular forces in a variety of cell types and tissues, and in orchestrating biological responses to these forces [5,6]. Nonetheless, our current understanding of how lung cells sense and respond to bidirectional forces is still very limited [1,7], due in part to the lack of suitable techniques to apply such forces to cells and to probe the corresponding cell mechanoresponses. There are several approaches to study local cell-ECM mechanical interactions either at the dorsal or ventral surface of adherent cells. These approaches have provided valuable insights into the complex nature of cell-ECM mechanical interactions. However, these approaches are subjected to limitations that undermine their application to study cell mechanoresponse to force directionality, including the inability to either apply both compression and extension to cells, to provide probe-independent cell mechanical responses due to undefined probe-cell contact geometry, or both [8,9,10,11,12,13]. A promising approach that may overcome these technical limitations is based on AFM provided with unconventional flat-ended cylindrical AFM tips (referred to as FE-AFM tips thereafter) [14]. Unlike standard sharp pyramidal AFM tips, FE-AFM tips provide well-defined contact geometry with the cell surface that remains constant and PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e32261