Exploring the Mechanical Properties of Single Vimentin Intermediate Filaments by Atomic Force Microscopy C. Guzmán 1 ⁎, S. Jeney 1 , L. Kreplak 2 , S. Kasas 3,4 , A. J. Kulik 1 U. Aebi 2 and L. Forró 1 1 Institut de Physique de la Matière Complexe, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland 2 M. E. Müller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstr. 70 4056 Basel, Switzerland 3 Institut de Neurosciences, EPFL, CH-1015 Lausanne, Switzerland 4 Institut de Biologie Cellulaire et de Morphologie, UNIL, CH-1005 Lausanne, Switzerland Intermediate filaments (IFs), together with actin filaments and micro- tubules, compose the cytoskeleton. Among other functions, IFs impart mechanical stability to cells when exposed to mechanical stress and act as a support when the other cytoskeletal filaments cannot keep the structural integrity of the cells. Here we present a study on the bending properties of single vimentin IFs in which we used an atomic force microscopy (AFM) tip to elastically deform single filaments hanging over a porous membrane. We obtained a value for the bending modulus of non-stabilized IFs between 300 MPa and 400 MPa. Our results together with previous ones suggest that IFs present axial sliding between their constitutive building blocks and therefore have a bending modulus that depends on the filament length. Measurements of glutaraldehyde-stabilized filaments were also performed to reduce the axial sliding between subunits and therefore provide a lower limit estimate of the Young’s modulus of the filaments. The results show an increment of two to three times in the bending modulus for the stabilized IFs with respect to the non-stabilized ones, suggesting that the Young’s modulus of vimentin IFs should be around 900 MPa or higher. © 2006 Elsevier Ltd. All rights reserved. *Corresponding author Keywords: vimentin; intermediate filaments; atomic force microscopy; bending stiffness; axial sliding between dimmers; glutaraldehyde-stabi- lized filaments Introduction For years it has been known that the cytoskel- eton, a complex network of filaments, plays a major role in determining cell architecture and mechanics. The dynamic interaction between its three components (intermediate filaments, micro- tubules and actin filaments) regulates to a great extent the structural organization of the cytoplasm of animal cells. 1 Intermediate filaments (IFs) are considered as the stress-buffering elements of metazoan cells 2 and it has also been demonstrated that they maintain the mechanical integrity essen- tial for cell migration, particularly during wound healing. 3–6 The superfamily of IF proteins, which includes at least 65 distinct proteins in humans, 2 has been divided into five different types according to their primary structure, gene structure, assembly proper- ties and their tissue-specific expression patterns regulated during development. 1 Structural studies have identified a common building block for all cytoplasmic IFs consisting of a very elongated rod-like dimer (45–50 nm) based on an α-helical coiled-coil structure. 7 This rod-like dimer exhibits a ‘‘tripartite’’ structure formed by an α-helical central rod domain and flanked by non- α-helical head and tail domains. The central rod is composed of four consecutive α-helical segments 1A, 1B, 2A and 2B that harbor a pronounced seven- residue periodicity, called heptad repeat, in the distribution of hydrophobic residues, 7 exhibiting the signature of a coiled-coil structure. The four α- helical segments are interconnected by short, vari- able linkers L1, L12 and L2 7 and have a structure and length that is highly conserved in vertebrate cytoplasmic IFs. 1 In contrast, the length and Abbreviations used: IF, intermediate filament; AFM, atomic force microscopy; ULF, unit-length filaments; MT, microtubule; TEM, transmission electron microscopy. E-mail address of the corresponding author: camilo.guzman@epfl.ch doi:10.1016/j.jmb.2006.05.030 J. Mol. Biol. (2006) 360, 623–630 0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.