FRET measurements of cell-traction forces and nano-scale clustering of adhesion ligands varied by substrate stiffness.

The mechanical properties of cell adhesion substrates regulate cell phenotype, but the mechanism of this relation is currently unclear. It may involve the magnitude of traction force applied by the cell, and/or the ability of the cells to rearrange the cell adhesion molecules presented from the material. In this study, we describe a FRET technique that can be used to evaluate the mechanics of cell-material interactions at the molecular level and simultaneously quantify the cell-based nanoscale rearrangement of the material itself. We found that these events depended on the mechanical rigidity of the adhesion substrate. Furthermore, both the proliferation and differentiation of preosteoblasts (MC3T3-E1) correlated to the magnitude of force that cells generate to cluster the cell adhesion ligands, but not the extent of ligand clustering. Together, these data demonstrate the utility of FRET in analyzing cell-material interactions, and suggest that regulation of phenotype with substrate stiffness is related to alterations in cellular traction forces.

[1]  M S Kolodney,et al.  Isometric contraction by fibroblasts and endothelial cells in tissue culture: a quantitative study , 1992, The Journal of cell biology.

[2]  O. Thoumine,et al.  Critical centrifugal forces induce adhesion rupture or structural reorganization in cultured cells. , 1996, Cell motility and the cytoskeleton.

[3]  Jon A. Rowley,et al.  Biomaterials to Spatially Regulate Cell Fate , 2002 .

[4]  J. Lakowicz.  Principles of fluorescence spectroscopy , 1983 .

[5]  Jennifer Linderman,et al.  Nanoscale Adhesion Ligand Organization Regulates Osteoblast Proliferation and Differentiation. , 2004, Nano letters.

[6]  E. Dill,et al.  An Introduction to the Mechanics of Solids , 1972 .

[7]  B. Geiger,et al.  Involvement of microtubules in the control of adhesion-dependent signal transduction , 1996, Current Biology.

[8]  A. Harris,et al.  Silicone rubber substrata: a new wrinkle in the study of cell locomotion. , 1980, Science.

[9]  R. Hochmuth,et al.  Mechanically stimulated cytoskeleton rearrangement and cortical contraction in human neutrophils. , 1995, Biophysical journal.

[10]  K. Beningo,et al.  Flexible substrata for the detection of cellular traction forces. , 2002, Trends in cell biology.

[11]  Viola Vogel,et al.  Fibronectin extension and unfolding within cell matrix fibrils controlled by cytoskeletal tension , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[12]  L. Addadi,et al.  Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates , 2001, Nature Cell Biology.

[13]  D J Mooney,et al.  Alginate hydrogels as synthetic extracellular matrix materials. , 1999, Biomaterials.

[14]  P. Krebsbach,et al.  Isolation and Characterization of MC3T3‐E1 Preosteoblast Subclones with Distinct In Vitro and In Vivo Differentiation/Mineralization Potential , 1999, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[15]  Daniel Choquet,et al.  Extracellular Matrix Rigidity Causes Strengthening of Integrin–Cytoskeleton Linkages , 1997, Cell.

[16]  Y. Wang,et al.  Cell locomotion and focal adhesions are regulated by substrate flexibility. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[17]  M A Horton,et al.  Single integrin molecule adhesion forces in intact cells measured by atomic force microscopy. , 1999, Biochemical and biophysical research communications.

[18]  J. Linderman,et al.  Externally Applied Cyclic Strain Regulates Localization of Focal Contact Components in Cultured Smooth Muscle Cells , 2002, Annals of Biomedical Engineering.

[19]  E O Pettersen,et al.  Cell adhesion force microscopy. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[20]  D. Ingber,et al.  Mechanical behavior in living cells consistent with the tensegrity model , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[21]  Eben Alsberg,et al.  Engineering growing tissues , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[22]  H. Saunders,et al.  Book Reviews : AN INTRODUCTION TO THE MECHANICS OF SOLIDS S. H. Crandall; N.C. Dahl; and T. J. Lardner McGraw-Hill Book Co. , New York, N. Y. (1972) , 1975 .

[23]  D. Ingber,et al.  Mechanotransduction across the cell surface and through the cytoskeleton , 1993 .

[24]  C M Lapiere,et al.  In vitro tubulogenesis of endothelial cells by relaxation of the coupling extracellular matrix-cytoskeleton. , 2001, Cardiovascular research.

[25]  Douglas A Lauffenburger,et al.  Co-regulation of cell adhesion by nanoscale RGD organization and mechanical stimulus. , 2002, Journal of cell science.

[26]  Christopher S. Chen,et al.  Cells lying on a bed of microneedles: An approach to isolate mechanical force , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[27]  Donald E. Ingber,et al.  The structural and mechanical complexity of cell-growth control , 1999, Nature Cell Biology.

[28]  D. L. Taylor,et al.  Traction forces of cytokinesis measured with optically modified elastic substrata , 1997, Nature.

[29]  Adam J. Engler,et al.  Myotubes differentiate optimally on substrates with tissue-like stiffness , 2004, The Journal of cell biology.

[30]  Kenneth M. Yamada,et al.  Transmembrane crosstalk between the extracellular matrix and the cytoskeleton , 2001, Nature Reviews Molecular Cell Biology.

[31]  P. Vos,et al.  Cell encapsulation: Promise and progress , 2003, Nature Medicine.

[32]  Kenneth M. Yamada,et al.  Taking Cell-Matrix Adhesions to the Third Dimension , 2001, Science.

[33]  M. Sheetz,et al.  A micromachined device provides a new bend on fibroblast traction forces. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[34]  Kenneth M. Yamada,et al.  Physical state of the extracellular matrix regulates the structure and molecular composition of cell-matrix adhesions. , 2000, Molecular biology of the cell.

[35]  M. Sheetz,et al.  Forces on adhesive contacts affect cell function. , 1998, Current opinion in cell biology.

[36]  Michael P. Sheetz,et al.  The relationship between force and focal complex development , 2002, The Journal of cell biology.

[37]  B. Herman,et al.  Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. , 1998, Biophysical journal.